Chemical and Biochemical Properties of Common Nettle (Urtica dioica L.) Depending on Various Nitrogen Fertilization Doses in Crop Production

Abstract

Fertilization in sustainable agriculture aims to provide optimal nutrients to plants while minimizing negative impacts on the environment and human health. This study aimed to determine the effect of various nitrogen fertilizer doses of 0, 50, 200, and 300 kgN ha⁻¹ on the chemical and biochemical composition of the leaves of nettle (Urtica dioica L.). Nettle leaves were harvested in late April to early May, before flowering. The contents of Zn, Cu, Mn, and Fe, as well as chlorophylls a and b, carotenoids, ascorbic acid, antioxidant, and catalase activity were determined. The result of catalase activity in nettle leaves was used to calculate the resistance index and the actual value of this enzyme activity, which was given as percentage change. Based on the analysis, nitrogen fertilization was found to have a statistically significant effect on the formation of the tested chemical and biochemical parameters in nettle leaves. The highest dose of nitrogen caused a statistically significant increase in the content of Zn, Mn, and assimilation pigments. The content of ascorbic acid ranged from 8.7 to 115 mg 100 g⁻¹ f.m. and, in contrast to the antioxidant and catalase activity, decreased with increasing nitrogen dose. The relative change index (RCh) showed the following effect of nitrogen dose on catalase activity: N300 > N200 > N50. The low value of the plant resistance index (RP) for the nitrogen dose of 300 kg N ha⁻¹ indicates that this dose had the greatest effect (lowest resistance) on catalase activity. Thus, the impact of the stress factor (nitrogen fertilization) was greatest at the highest dose. No statistically significant differences in catalase activity were found between N0 and N50. It was also demonstrated that the contents of Zn and Mn had a statistically significant and positive correlation with chlorophyll a and chlorophyll b.

1. Introduction

The primary goal of fertilization in sustainable agriculture is to meet the nutritional needs of plants at a level that allows for profitable, high-quality yields and reduces threats to the natural environment. For nitrogen fertilization to be effective, the remaining elements of sustainable agriculture should be at the right level. Incorrect fertilization of plants affects their reactions and defence mechanisms against abiotic stress, which ultimately affects crop production [1]. Nitrogen (N) is a nutrient necessary for plant growth and development. This element can improve nutrient uptake, enzyme activity, and photosynthesis rate [2,3]. N deficiency in soil accelerates leaf yellowing through chlorophyll degradation, which reduces the rate of CO₂ assimilation and decreases protein content [4]. Its excess or deficiency in soil also causes oxidative stress and the production of reactive oxygen species (ROS) [5]. Among other things, this leads to changes in the content of chlorophyll and nucleic acids in crop plants. According to Yang et al. [6], N is used for chloroplast synthesis in leaves, and the application of nitrogen fertilizers can simulate chlorophyll synthesis. However, their excessive agricultural use poses serious problems, such as nitrogen oxide emission and groundwater pollution with nitrate nitrogen [7].

Nettle is a plant that has been widely exploited over a long period. The main herbaceous raw materials are the leaves (Urticae folium), which contain biologically active compounds with health-promoting properties [8]. The presence of valuable biological compounds, such as proteins, vitamins, phenolic components, macro- and micronutrients, tannins, flavonoids, sterols, fatty acids, carotenoids, and chlorophylls, contribute in various ways to the use of nettle. Its high nutritional value has led to nettle leaves being consumed by humans directly and as an animal feed [9,10]. Nettle leaf extract is used in the manufacture of personal care and pharmaceutical products and in herbal medicine [11,12]. Nettle is valued in part for its high content of mineral substances, including micronutrients, especially iron [13,14,15]. Nettle herb or leaves increase the level of haemoglobin and red blood cells; these effects are attributed to the haematopoietic effect of iron. The leaves also have high concentrations of some rare elements, such as titanium [16] and trace amounts of essential oil (anthophene) [17]. Chlorophyll a and b are obtained from nettle leaves and are widely used in cosmetics, food, and medicine [18]. In the food industry, it is known as the colouring E140 [19]. Chlorophyll content is also an indicator of plant response to anthropogenic, soil, and climatic conditions [20]. Carotenoids are the main source of provitamin A [21].

Thus far, nettle leaves have been gathered from natural places. However, the plant’s ability to accumulate heavy metals may imply quality problems in the raw material in wild specimens [22]. The growing demand for nettle leaves is requiring larger-scale cultivation. Nettle grows best in loose, nitrogen-rich soil, which is why the plant is described as nitrophilic. The cultivation instructions [23] recommend the following fertilizer doses: N 60–90 kg ha⁻¹, P₂O₅ 50–60 kg ha⁻¹, K₂O 80–100 kg ha⁻¹. However, data in the literature provide different nitrogen doses for nettle cultivation: 160–300 kg ha⁻¹ [24], 300–400 kg ha⁻¹ [25], and even 440 kg ha⁻¹ [26]. Martinez-Ballesta et al. [27] found that applying high doses of nitrogen improved the growth and yield of nettle, but high concentrations may pollute the environment and cause accumulation in leaves. Another significant factor regulating plant growth and the quality of nettle raw material is the form of nitrogen [28]. The content of health-promoting compounds in plants is regulated by minerals in the soil.

Plants employ non-enzymatic and enzymatic mechanisms to adapt to conditions, especially stress conditions. The non-enzymatic mechanisms include pigments and vitamins (e.g., carotenoids, ascorbate, chromanols, flavonoids, glutathione, ubiquinol). The enzymatic mechanisms include catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. These are enzymes that directly remove ROS [29]. Catalase is one of the most important enzymes involved in defending organisms against the negative effects of oxidative stress. Catalase catalyses the decomposition of harmful hydrogen peroxide into water and oxygen [30].

The aim of the study was to evaluate the effect of nitrogen doses on the content of micronutrients and selected non-enzymatic and enzymatic antioxidants in nettle leaves.

2. Materials and Methods

2.1. Soil Sampling Location

The research area was located in a heavily agricultural part of the Kuyavian-Pomeranian Voivodeship in north-central Poland (52°49′02″ N 17°34′25″ E). The study area is located in the eastern part of the Chodzież Lakeland, between the middle Noteć valley and the Wełna valley with the right tributary of the Warta river Kondracki 2012 [31]. The annual sum of precipitation in this region is 668 mm, and the average annual temperature is 8.8–9.3 °C (Figure 1) (average from 2021–2023).

The study area is dominated by Luvisols [32]. The research was conducted in 2021–23. The article presents average research results for the years 2022 and 2023. This averaging was intended to eliminate the influence that such abiotic factors as precipitation or temperature might have had on the tested parameters.

The single-factor experiment was established using 10 to 15 cm-long stolon cuttings in the autumn of 2021 in three replicates, with harvest plots of 1 m². Nettle rhizomes were planted in furrows spaced every 35 cm. During the course of the plant growing period, the nettle was weeded and watered as required. Fertilization in the form of ammonium nitrate (NH₄NO₃) was applied in doses of 0 (control), 50, 200, and 300 kg N ha⁻¹. Mineral fertilizers (P and K) were applied in a single dose of 70 kg P₂O₅ ha⁻¹ and 100 kg K₂O ha⁻¹ before establishing the experiment and, in the following years, before the onset of vegetation. Common nettle was harvested before flowering (April). After harvesting, stems were separated from leaves. The samples were stored at −18 °C and lyophilized.

2.2. Method

2.2.1. Soil Analysis

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Physicochemical properties were determined in samples of disturbed soil, taken from a horizon of 0–25 cm. The soil was air-dried, sieved through a 2 mm mesh, and granulometric composition determined by laser diffraction using a Mastersizer MS 2000 analyser (Malvern Panalytical, Worcestershire, UK)

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pH was measured potentiometrically in 1 M KCl extract [33]

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Total organic carbon (TOC) and total nitrogen (TN) were determined with the Vario Max CN analyser (Elementar, Hanau in Germany).

2.2.2. Plant Analysis

The lyophilized nettle leaves were crushed and subjected to wet mineralization in concentrated H₂SO₄ with the addition of H₂O₂.

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The contents of Zn, Cu, Mn, and Fe in the extract were determined by flame absorption spectroscopy using a Solaar S4 apparatus (Thermo Elementar, Germany).

The nettle leaves were also subjected to biochemical analysis. The contents of plant metabolites (chlorophyll a, chlorophyll b, carotenoids, and ascorbate) as well as catalase activity and antioxidant activity were determined according to the following procedures:

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The contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids were determined according to Lichtenthaler [34] as well as Lichtenthaler and Buschmann [35]. The contents of chlorophyll a, chlorophyll b, and carotenoids were determined using a spectrophotometer at wavelengths (λ_max) of 645 nm, 662 nm, and 470 nm, respectively. The content of plant pigments was calculated in milligrams per gram of fresh weight of the sample (mg g⁻¹ f.w.). The contents of Chl a and Chl b were used to determine the ratio of these pigments (Chl a/b, which is an indicator of leaf health) and the total chlorophyll content (a + b).

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The content of ascorbic acid (AAC) was determined by titration in an acidic medium with a solution of the standard dye 2,6-dibromophenolindophenol until a pink colour was obtained for 30 s [36].

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Antioxidant activity (AA) was determined using the 2,2-diphenyl−1-picrylhydrazyl free radical (DPPH) method according to Zeipin et al. [37]. To 1 g of nettle leaves, 10 mL of methanol was added. After adding the methanolic DPPH solution, the spectrophotometric measurement was performed at a wavelength of 515 nm. Antioxidant activity (%) was calculated by the following equation:

$$AA=100\times \left(1-{\displaystyle \frac{A_{SS}}{A_0}}\right)$$

(1)

where Ass—absorbance of the test sample; A₀—absorbance of the control DPPH solution with 0.3 mL of methanol.

Catalase (CAT) activity in nettle leaves was determined by the method described by Kar and Mishra [38] with H₂O₂ as substrate. The residual H₂O₂ was titrated against 0.01 N KMnO₄ until a faint purple colour persisted for at least 15 s. Based on the results of catalase activity, the plant resistance index (RP) was calculated using the formula proposed by Orwin and Wardle [39]:

$$RP=1-\left[{\displaystyle \frac{2\left|D_0\right|}{\left(C_0+\left|D_0\right|\right)}}\right]$$

(2)

The RP index value is delimited by −1 and +1. A value of 1 means that N fertilization had no effect on catalase activity (maximum resistance). The lower the value, the stronger the effect of N on catalase. An RP value of 0 indicates 100% inhibition of catalase activity.

The actual value of catalase activity was also calculated and given as percentage change (relative change) in relation to the control plant [40]:

$$RCh=\left({\displaystyle \frac{P_0}{C_0}}-1\right)\times 100$$

(3)

where for both indicators: $D_0=C_0-P_0$, $C_0$—catalase activity in the leaves of the control nettle (N0), $P_0$—catalase activity in the leaves of nettle fertilized with N (N50–N300).

2.3. Statistics

Based on the results, statistical analysis was performed using STATISTICA 13 (Stat Soft Polska). A one-way analysis of variance (ANOVA) was performed for a randomized block design to determine the effect of nitrogen dose on the chemical and biochemical parameters of the nettle leaves. Means with standard deviation (±SD) were calculated. Tukey’s post hoc test was used to identify statistically significant differences between means. All chemical and biochemical analyses were performed in triplicate. The results were presented as arithmetic means. In this study, Pearson correlation coefficients between the studied parameters were also calculated using the PAST 4.13 program [41]. Only statistically significant correlation results are presented, in the form of a correlogram.

3. Results and Discussion

The analysed soils had a highly homogeneous granulometric composition. All soil samples were classified into one soil texture class “sandy loam” [42]. The soil material was dominated by sand and silt fractions. The clay content ranged from 6.10 to 6.28%.

The pH values of the tested soils ranged from 5.44 in the soil fertilized with the highest dose of nitrogen to 6.63 in the control soil (

Table 1. Selected physicochemical properties in soil.

Dose Nitrogen kg N ha −1	Sand %	Silt %	Clay %	pH 1M KCl	TOC g kg−1	TN g kg−1
* N0	55.66 ± 5.31	38.24 ± 4.40	6.10 ± 0.16	6.63 ± 0.14	12.46 a ± 0.03	1.20 c ± 0.02
N50	53.82 ± 4.50	40.05 ± 3.92	6.13 ± 0.12	5.88 ± 0.22	12.30 ab ± 0.12	1.33 b ± 0.01
N200	55.04 ± 5.66	38.68 ± 5.23	6.28 ± 0.12	5.61 ± 0.18	11.66 c ± 0.17	1.38 b ± 0.02
N300	55.84 ± 4.82	37.99 ± 4.80	6.17 ± 0.07	5.44 ± 0.10	12.17 b ± 0.08	1.69 a ± 0.04

* N0—0 kgN ha⁻¹; N50—50 kgN ha⁻¹; N200—200 kgN ha⁻¹; N300—300 kgN ha⁻¹. Different small letters indicate significant differences between levels of the experience factor (nitrogen dose).

). Agricultural mineral fertilizers, especially nitrogen ones, increase soil acidification [3]. Table 1 presents TOC and TN contents. The TOC content was highest in the control soil samples at 12.46 g kg⁻¹, and lowest in the soil fertilized with the N200 dose at 11.66 g kg⁻¹. TN content was inversely proportional to TOC content. The TN content had the highest statistically significant value in soils fertilized with the N300 dose at 1.69 g kg⁻¹, and the lowest in the control soil.

The ratio of TOC to TN in sustainable agriculture is an important indicator of soil quality and its ability to provide nutrients to plants. The TOC/TN ratio in the soil influences nitrogen mineralization and immobilization processes. High values lead to nitrogen immobilization, i.e., nitrogen being bound by microorganisms and becoming temporarily unavailable to plants. In turn, appropriate nitrogen fertilization helps to narrow this ratio and release nitrogen, accelerating the decomposition of organic matter [43]. The control soil also showed a higher statistically significant TOC/TN ratio (Figure 2).

Nettle is a valuable source of macro and micronutrients. The literature reports contents at various levels: Zn 12.70–48.40 mg kg⁻¹, Cu 10.23–18.43 mg kg⁻¹, Mn 30.50–412 mg kg⁻¹, Fe 112.60–526.20 mg kg⁻¹ [12,14,44]. The concentration of mineral components in nettle varies greatly due to differences in contents of minerals taken from the soil and due to the development phase and harvest date of the plant [12,13,45]. The content of micronutrients in the analysed nettle leaves was Mn > Fe > Zn > Cu (

Table 2. The contents of Zn, Cu, Mn, and Fe in nettle leaves as a function of nitrogen doses.

Nitrogen Dose kg N ha−1	Zn	Cu	Mn	Fe
	mg kg−1
* N0	20.27 b ± 2.15	6.69 a ± 1.05	376.2 c ± 3.34	305.2 ab ± 9.70
N50	20.55 b ± 3.09	6.08 ab ± 1.99	417.5 bc ± 19.26	368.8 a ± 10.15
N200	24.98 a ± 1.28	5.75 b ± 2.17	553.6 ab ± 8.84	265.5 b ± 3.75
N300	25.48 a ± 2.42	4.22 c ± 1.44	634.5 a ± 11.92	340.6 a ± 21.29

* see Table 1.; different small letters indicate significant differences between levels of the experience factor (nitrogen dose).

). The manganese content in nettle leaves ranged from 376.2 to 634.5 mg kg⁻¹ and was dependent on nitrogen doses, increasing with the dose. Similar relationships were found by Szewczuk et al. [13]. With increasing N doses, they noted a gradual increase in the concentration of the element in both stems and leaves. However, differences in nitrogen fertilizer doses did not consistently affect the content of Fe. Iron contents ranged from 265.5 to 368.8 mg kg⁻¹. The doses of nitrogen fertilizers applied did not directionally modify the concentration of this component. Rodman et al. [15] found that nitrogen fertilization negatively affected the iron content in nettle plants, but this content increased as yields increased. The highest zinc content in nettle leaves was found in the plots fertilized with the highest doses of nitrogen. The highest statistically significant contents of this element were recorded with fertilization doses of N300 and N200, being, respectively, 25.48 mg kg⁻¹ and 24.98 mg kg⁻¹. The copper content in plants followed an inverse pattern. When fertilized with N300, the copper content in plants was lowest at 4.22 mg kg⁻¹. The highest statistically significant content of this element was found in plants not fertilized with nitrogen (Table 2). Martinez-Ballesta et al. [46] found the effect of nitrogen fertilizers on the content of micronutrients in nettle plants to depend on dose, micronutrient tested, species, plant part, and developmental stage. According to Rutto et al. [47], producers can manage the quality and quantity of nettle yield through judicious fertilizer supply, planting time, and the selective harvesting of different plant parts.

The source of micronutrients in plants is their accumulation through the root system from the soil, which is characteristic for a given plant and distinguishes individual plant habitats. The concentration of mineral components in nettle may vary greatly due to differences in uptake from the soil among the nutrients [12,14]. Statistically significant in the process of metal migration from the soil solution, where they are easily available to plants, is the role played by changes in pH, redox potential, temperature fluctuations, and the decomposition of organic matter or mycorrhiza [48,49,50]. In our own studies, correlation analysis confirmed the statistically significant influence of soil reaction and TOC on the content of the micronutrients tested in the plant (Figure 3). Also, the species-specific plant mechanism of uptake through roots may influence the relationship between the concentration of elements in the soil and the actual concentration in the leaves. Boshoff et al. [51] found that species-specific features of nettle (extensive root system) and uptake mechanisms may play a very important role in the accumulation of nutrients by plants.

Sustainable nitrogen fertilization has a significant impact on the chlorophyll content of plants. Nitrogen is a key component of chlorophyll. Chlorophylls a and b are the main photosynthetic pigments of seed plants. Together with carotenoids, they are part of the photosystems. In the human diet, they also exhibit anti-obesity, antioxidant, and anti-cancer effects [52]. The results of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) contents are presented in

Table 3. Content of chlorophyll a, b, and carotenoids in nettle leaves as a function of nitrogen doses.

Nitrogen Dose kg N ha −1	Chl a mg g−1 FW	Chl b mg g−1 FW	Car mg g−1 FW
* N0	0.641 c ± 0.021	0.256 c ± 0.022	0.215 d ± 0.015
N50	0.651 c ± 0.019	0.266 c ± 0.021	0.561 c ± 0.055
N200	1.227 b ± 0.063	0.396 b ± 0.035	0.952 b ± 0.074
N300	1.936 a ± 0.088	0.659 a ± 0.070	1.231 a ± 0.086

Chl a—chlorophyll a; Ch b—chlorophyll b; Car—carotenoids. * see Table 1.; different small letters indicate significant differences between levels of the experience factor (nitrogen dose).

. Analysis of variance showed that the experimental factor used (N doses) had a statistically significant effect on the content of the tested plant pigments. The content of Chl a, Chl b, and Car had the highest statistically significant value in nettle leaves grown at the highest nitrogen dose N300, being, respectively, 1.936, 0.659, and 1.231 mg g⁻¹ FW. This was, respectively, 302%, 257%, and 574% of the level for N0. The trend for Ch a and Ch b content was N300 > N200 > N50 = N0. The same trend for Car content was N300 > N200 > N50 > N0. Mohammad et al. [1] found that nitrogen has a fundamental impact on increasing leaf area and thus on the photosynthesis process. Higher N content in the soil protected the photosynthetic system. The study by Peng et al. [53] showed that the content of Chl a and Chl b in rice leaves at the germination, earing, and ripening stages increased as nitrogen application increased. This indicates that nitrogen deficiency can lead to a decrease in chlorophyll content.

The highest statistically significant chlorophyll a/b ratio value (3.10) was obtained in nettle leaves fertilized with N200 (Figure 4a). According to Sonobe et al. [54], values above 3 indicate stress under nitrogen deficiency conditions. Our research results did not confirm this relationship, probably because nettle is nitrophilic. For the remaining N doses, the chlorophyll a/b ratio was statistically significantly lower and ranged from 2.45 to 2.93. A higher chlorophyll a/b ratio indicates greater peroxidation activity in leaf membranes. The content of total chlorophyll (chlorophylls a + b) had the highest statistically significant value after the application of the N300 dose (Figure 4b). No statistically significant differences were found for chlorophylls a+b at doses N0 and N50. The application of N fertilization resulted in a statistically significant decrease in the chlorophylls-(a + b)-to-carotenoid ratio (Figure 4c). Changes in the chlorophylls-(a+b)-to-carotenoids ratio [54] can be used to assess environmental stress in plants.

Ascorbic acid (AAC) is one of the non-enzymatic antioxidants that protects plants against oxidative damage by scavenging free oxygen radicals [55]. The AAC content in nettle leaves ranged from 8.7 to 115 mg 100 g⁻¹ FW. (

Table 4. Content of ascorbic acid, antioxidant, and catalase activity as a function of nitrogen doses.

Nitrogen Dose kg N ha −1	AAC mg 100 g−1 FW	AA %	CAT mg H2O2 kg−1 h−1
* N0	115.0 a ± 1.53	54.79 c ± 1.17	15.87 c ± 0.846
N50	87.4 b ± 1.11	58.12 c ± 1.53	16.22 c ± 0.952
N200	12.3 c ± 0.863	67.56 b ± 1.89	21.96 b ± 1.09
N300	8.7 c ± 0.258	82.35 a ± 2.56	29.85 a ± 1.23

AAC—ascorbic acid content; AA—antioxidant activity; CAT—catalase. *see Table 1.; different small letters indicate significant differences between levels of the experience factor (nitrogen dose).

) depending on the experiment used. According to the literature [12,44], ascorbic acid (AAC) content can range from 16.00 to 112.8 mg 100 g⁻¹ FW or even up to 238 mg 100 g⁻¹. The highest statistically significant content of ascorbic acid was found at N0 (115 mg 100 g⁻¹ FW), and increasing N doses had a negative effect on the ascorbic acid content. A similar tendency was noted in the study by Radman et al. [14]. Unbalanced N fertilization can disrupt plant metabolism, leading to reduced ascorbic acid synthesis. According to Rajasree and Pillai [56], there is an inverse relationship between ascorbic acid content and nitrogen. Higher N doses cause excessive vegetative growth of plants and shading, which reduces the ascorbic acid content. Increased N content in soil also increases the respiration rate, which affects the concentration of ascorbic acid in the cell, either increasing its consumption rate or decreasing its synthesis.

Antioxidant activity (AA) is the ability of a substance to neutralize free radicals and other reactive molecules that can damage cells. The AA of nettle leaves ranged from 54.78% to 82.35% (Table 4). According to Zeipiņa et al. [37], antioxidant activity in nettle leaves ranged from 17.31 to 80.77%, depending on soil fertility. This was confirmed by our studies, in which N fertilization had a statistically significant effect on AA. Nettle leaves fertilized with an N dose of N300 were characterized by the highest statistically significant AA (82.35%) value. No statistically significant differences in AA were found between N0 and N50. Optimal nitrogen fertilization helps plants cope with oxidative stress by providing essential nutrients and supporting defence systems. According to Caballero et al. [57], the higher antioxidant activity of nettle leaves is attributed to their chlorophyll, β-carotene, ascorbic acid, manganese (Mn), and zinc (Zn) content. These compounds are nutritional antioxidants.

Catalase activity is one of the basic indicators determined to assess oxidative stress in plants [58]. The results showed that CAT activity increased with increasing N dose (Table 4). No statistically significant differences in CAT activity were found between N0 and N50. Muhammad et al. [1] found that the higher enzymatic activity of antioxidants, including catalase, increases yields by protecting the photosynthetic system of plant cultivation. N application reduced the H₂O₂ and O₂ content in the plant. This is probably associated with N potentially being beneficial in maintaining aquaporin activity by reducing H₂O₂ accumulation [1]. Noor et al. [59] found a statistically significant increase in CAT activity after applying a nitrogen dose of up to 150 kg ha⁻¹. A dose of 210 kg ha⁻¹ caused a decrease in CAT activity. However, it had a higher statistically significant value compared to N0 and N120. In this case, wheat was used for the research. Research by Ahmad et al. [60] showed that, with maize, an optimal level of N fertilization significantly increased the activity of antioxidant enzymes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase) while also reducing the aging of leaves. However, further increases in N doses adversely affected the activity of these enzymes.

The RP values presented in Figure 5a indicate that the sensitivity of catalase to the application of nitrogen was dose dependent. The RP value was significantly highest for N0 (0.921) and lowest for N300 (0.063). Higher RP index values indicate that nitrogen had little effect (maximum resistance) on catalase activity and consequently the stress factor had the smallest impact. The RP resistance index is the effective response of enzymes to environmental stress [39,61]. Adding nitrogen to the soil activated catalase activity in nettle leaves (positive RCh values). The percentage changes in RCh indicate the direction of the experimental factor’s action (inhibition or activation) on enzymatic activity [40,62]. The applied doses of nitrogen activated catalase in nettle leaves (Figure 5b). Based on the RCh coefficient value, the effect of nitrogen doses was N300 > N200 > N50.

The correlation analysis showed a statistically significant positive correlation between the AA of nettle leaves and the contents of Chl a (r = 0.989), Chl b c (r = 0.979), and Car (r = 0.953) (Figure 3). The study by Paulauskienne et al. [12] showed that AA correlated moderately positively with Chl a (r = 0.67) and Chl b (r = 0.56). A negative correlation was found between the enzymatic coefficients RP and RCh (r= −0.412). Increased activation of catalase (RCh) in nettle leaves under the N300 dose resulted in lower resistance of this enzyme.

The study showed that contents of Zn and Mn had a statistically significantly positive correlation with Chl a (r = 0.912; r = 0.904, respectively) and Chl b (r = 0.816; r = 0.904) (Figure 3). Micronutrients play a key role in the content and production of chlorophyll in plants. Several microelements are essential for chlorophyll biosynthesis, including iron, magnesium, manganese, and zinc. These elements contribute to various enzymatic reactions and structural components needed for chlorophyll formation, influencing photosynthesis and overall plant growth [63,64].

One indicator of the quality of crops is the content of ascorbic acid (AAC). Bhusal et al. [65] and Feszterova et al. [66] found that nettle leaves have a high nutritional value, which indicates their potential as a source of vitamin C. The AAC content in the plants is particularly influenced by Cu, Ni, Mn, Zn, and Bo [67,68]. This paper found that AAC had a statistically significantly negative correlation with Zn (r= −0.983) and Mn (r = −0.939). However, a positive correlation was found between AAC and Cu (r = 0.809) (Figure 3). According to Mishra et al. [69], there is a complex correlation between vitamin C content in plants and micronutrients (Zn, Cu, Fe, and Mn). Zinc plays a role in various metabolic and physiological processes, and a deficiency of it affects the level of AAC. Zinc deficiency may affect vitamin C levels. Manganese can increase vitamin C content in plants. Iron is involved in the transport and utilization of other nutrients, which can affect the synthesis of vitamin C. Copper deficiency can lead to changes in vitamin C content, potentially affecting antioxidant capacity [69]. Bodo et al. [70] found a statistically significant relationship between the content of elements, especially zinc, and the antioxidant properties of honey. Zinc and, even more so, selenium are micronutrients with confirmed antioxidant properties [71,72]. In our research we found positive correlations between CAT activity and the contents of Zn (r = 0.912) and Mn (r = 0.935). No relationship was found between CAT and Fe. Catalase is a metalloenzyme (a haemoprotein—contains iron at the active site). Plants with low Fe content may show higher CAT activity than those with sufficient Fe, probably as a compensatory mechanism. Catalases can also occur in a form in which manganese is present at the active site instead of iron. One of the biological functions of Mn is its association with oxygen metabolism, as a cofactor of antioxidant defence enzymes [73].

4. Conclusions

Increasing doses of nitrogen fertilizers directionally modified the content of Mn, Zn, and Cu in nettle leaves, but did not affect the content of Fe. The high variability in the content of the tested microelements in the plant may result from differences in the contents of these elements in the soil, as well as from soil reaction, which strongly affects their absorption.

The content of assimilation pigments in nettle leaves increased with increasing nitrogen doses.

The content of ascorbic acid as a non-enzymatic antioxidant was lowest in nettle leaves collected from the experimental variants in which a dose of 300 kg N ha⁻¹ was applied. By contrast, antioxidant activity and catalase activity were highest in this variant. In terms of RCh coefficient values for catalase activity in nettle leaves, the effect of nitrogen doses was N300 > N200 > N50. The impact of nitrogen fertilization as a stress factor for catalase activity was the highest for the nitrogen dose of 300 kg ha⁻¹, as evidenced by the low value of the plant resistance index (RP).