Effect of Foliar Application of Iron Oxide Nanoparticles on Some Plant Enzymes of Chamomile (Matricaria Chamomilla L.) Under Different Irrigation Levels

INTRODUCTION
German chamomile Matricaria chamomilla L. has been known as a popular medicinal plant that has been widely used in traditional and folk medicine today. Therapeutic, cosmetic and nutritional benefits backed by years of traditional practices as well as scientific inquiry [1]. A well-known medicinal and herbal remedy in the Asteraceae (Compositae) family being common, its various medicinal and aromatic properties are referred to as the “Star of Medicinal Plants [2].
However, its cultivation is severely hampered by environmental stresses. One of the most harmful abiotic stresses is salinity, which drastically lowers agricultural productivity and quality globally. Currently, salt stress affects more than 20% of all arable land on Earth, and this percentage is rising as a result of both natural and human-caused factors. Through a variety of mechanisms, such as nutritional imbalances, hormonal disruptions, ionic toxicity, osmotic stress, and oxidative damage, salinity negatively affects plant growth and development. As a result, these physiological disruptions make plants more vulnerable to certain illnesses [3]. As the world’s population grows, agricultural nanotechnology has become a useful tool for increasing crop yields and agricultural productivity. The use of nanoparticles (NPs) as bio-stimulants or nano-fertilizers has grown dramatically over the last ten years to enhance the morphological and biochemical characteristics of a variety of crops, especially in agricultural areas without sustainable management techniques. [4].
This study’s main goal is to assess how foliar application of iron oxide nanoparticles (Fe2O3 NPs) affects particular plant enzymes in chamomile Matricaria chamomilla L. at different salt stress levels.

MATERIALS AND METHODS
A pot experiment was conducted during the winter season of 2025–2026, following a four-replicate completely randomized design (C.R.D.). There were two primary components to the experiment:
1. Foliar Application of Fe2O3 NPs: Four concentrations were applied (0, 25, 50, and 100 mg/L-1).
2. Salinity Stress: Four levels of saline irrigation were utilized (2, 4, 6 and 8dS/m-1)
The treatments were applied over four stages, beginning at the 4–6 leaf stage. Seeds were sown on October 15, 2025, i15 kg plastic pots are available. Firstly, ten seeds were planted per pot, and seedlings were thinned to four plants per pot 20 days post-planting. Soil samples were collected prior to cultivation for baseline characterization (Table 1). Nano-iron oxide solutions were prepared according to the required concentrations, The nano-iron oxide (Fe2O3 NPs) solutions were prepared according to the predetermined concentrations. The specific weight of the (Fe2O3) nanoparticles (powder form) was added to a measured volume of distilled water. The mixture was thoroughly agitated (Shaken) to ensure proper dispersion and reach the target concentration.
To enhance the efficacy of the foliar treatment, a commercial surfactant (detergent) was added to the solution as a spreading agent to ensure maximum wetting of the vegetative parts. Foliar spraying was conducted during the early morning hours using a 3-liter manual sprayer until the foliage was completely saturated. For the control group, Only distilled water was used to shower the plants. There were 64 experimental units in the experimental setup.

Estimation of catalase enzyme (CAT) Activity (katal / g f.wt)
The following equation was used to calculate CAT activity using the method outlined by [5]:

Catalase Activity of Test kU= (2.303/t) × log So/S

Estimation of Superoxide Dismutase (SOD) Activity (U/g.f.wt)
The following formulas were used to calculate SOD activity in accordance with the approach of [6]:
ΔA control - ΔA test

% Inhibition of pyrogallol autoxidation = X 100%ΔA control

(Cu-Zn) SOD Activity (U/ml) = % inhibition of pyrogallol autoxidation/50%

Estimation of Peroxidase (POD) Activity (U/g.f.wt)
POD activity was calculated using the following equation:

∆ A420/20sec x Vt x dilution factor 

Volume activity (U/ml) = Vs x 12

Where, Vt = Total reaction volume (3.0 ml)., Vs = Total sample volume (0.10 ml), 12 = A constant representing the absorbance of 1 mg/ml solution of purpurogallin at 420 nm, Volume activity (U/ml) = ∆ A420 / sec×2.5 × dilution factor.

Estimation of Ascorbate Peroxidase (APX) Activity (U/g.f.wt})
APX activity was determined using the following formula:

APX activity (U/g tissue) = [(△A/Ɛ*1) ÷ t]*D.F

APX activity (U/g tissue) = Enzyme activity, △A: Change in absorbance over time, Ɛ: Molar extinction coefficient, D.F: Dilution Factor., (Ɛ = extinction coefficient = 2.8 mm-1 cm-1).

RESULTS AND DISCUSSION
Catalase (CAT) Enzyme Activity (katal / g. f.wt)
Table 2 findings show a considerable impact of foliar application with iron oxide nanoparticles (Fe2O3 NPs) on the leaf catalase (CAT) content of chamomile Matricaria chamomilla L. under the various levels of salinity stress investigated in this study:
The results indicated a significant impact of irrigation water salinity levels on the leaf catalase (CAT) content. Increased salinity led to a corresponding rise in CAT activity, reaching 0.2073, 0.2445, and 0.2596 katal/g f.wt at salinity levels of 4, 6, and 8 dS/m-¹, in contrast to 0.1597 katal/g f.wt at the control salinity level of 2 dS/m-¹.
The increase is due to the fact that high salt concentrations maintain the plant under environmental pressure, which causes an excess of H2O2 and O- reactive oxygen species (ROS). Antioxidant enzymes act to reduce these deleterious substances, and CAT, which cleaves H2O2 to O2 and H2O, protects the cell from oxidative injury [7]. These results are in line with those of [8,9], who found an increase rate of 18.25% at salinity levels of 3 and 6 dS/m-¹.
The results in the aforementioned table further indicate that foliar application of iron oxide nanoparticles (Fe2O3 NPs) had a significant effect on leaf CAT content. The activity increased to (0.1765, 0.1852, and 0.3403) (katal/g. f.wt) compared to the control treatment, which recorded 0.1611(katal/g. f.wt)
This enhancement is due to the role of nano-iron in stimulating the antioxidant defense system and reducing the accumulation of ROS [10]. These results align with the findings of [11] in their study on Arachis hypogaea, where an increase in CAT activity was observed at a concentration of 50 mg/L. However, they contrast with [12] who reported a decrease in CAT concentration at 400 mg/L compared to the 200 mg/L treatment of iron oxide nanoparticles in Carum copticum L.
The leaf catalase enzyme concentration was significantly impacted by the binary interaction between the study conditions, irrigation water salinity and foliar spraying with nano-iron oxide. Treatment F3S3 had the greatest value, 0.4695 (g.f.wt), while treatment F3S3 had the lowest value F0S0, which reached 0.1354 (g.f.wt). This is attributed to the fact that iron activated the defense system and increased catalase enzyme activity to reduce the damage resulting from salinity [13]. This is consistent with the findings of [14] upon applying nano-iron to Zea mays L. plants, as it mitigated salinity stress and increased catalase enzyme activity by a rate of 48.5%.

Superoxide dismutase enzyme (SOD) (u/g.f.wt)
The findings displayed in Table 3 demonstrated a noteworthy impact of irrigation water salinity levels on the superoxide dismutase (SOD) enzyme content. Increased salinity levels led to an increase in SOD enzyme activity, which rose to (0.273, 0.285 and 0.322) (u/g.f.wt) at salinity levels of (4, 6, and 8 dS m-1) respectively, compared to the irrigation water level of 2 (dS m-1), which reached 0.261 (u/g.f.wt). The explanation is that the plant’s physiological functions, including respiration and photosynthesis, are disrupted when it is exposed to salinity, leading to the formation of reactive oxygen species (ROS) such as the superoxide ion {O2-} and hydrogen peroxide {H2O2}. These compounds are toxic to cells, and the plant, as a defense mechanism, produces the SOD enzyme, which in turn converts the {O2-} ion into hydrogen peroxide {H2O2} and oxygen. After that, other enzymes like catalases and peroxidases act on the degradation of the hydrogen peroxide [15]. Overall, this is similar to the observations made by [16] by [17] 7.5 and 15 dS m-1 salinity level in Phaseolus vulgaris plants.
The data obtained in the above table indicated that foliar spraying of plants with nano-iron oxide had a significant effect on the leaf superoxide dissipation (SOD) enzyme content in compared to the control treatment (0.274 (u/g.f.wt)) and increased it to ( 0.284, 0.286,and 0.298) (u/g.f.wt). This is because the augmenting effects of nano-iron regulate the defence system, increase the SOD enzyme activity and reduce the oxidative stress in plants [10]. These results are consistent with Singh et al. who reported in Eucalyptus tereticornis plants. found that the activity of this enzyme in response to nano-iron oxide at a concentration of 25 mg L-1 increased to a 3.8-fold (3.8 times) of control. The concentration of the leaf superoxide dismutase (SOD) enzyme was greatly modified by the binary interaction of the two experimental factors of this study، irrigation water salinity and foliar spraying with nano-iron oxide. F2S3 treatment (0.445 (u/g.f.wt)) and F0S3 treatment (0.153 (u/g.f.wt)) showed the highest and lowest values of (u/g.f.wt), respectively. This is due to the fact nano iron increased the growth of the plant, reduced oxidative stress, and played a role in the stimulation of SOD enzyme activity [17]. Such is the case with the results obtained from the SOD enzyme in Rubus baileyanus plants, in which case the highest value was in concentrations of 2.8(mg L-1) of nano-iron at a salinity level of 3.8 (dS m-1).

Peroxidase Enzyme (POD) (u/g.f.wt)
The peroxidase (POD) enzyme concentration (u/g.f.wt) was significantly impacted by irrigation water salinity levels, according to the results displayed in Table 4. The POD enzyme increased as salt levels rose, reaching (225.7, 270.0, 353.7)(u/g.f.wt) at salinity levels of (4, 6, 8) (dS.m-1), respectively, compared to the irrigation water level of 2 dS.m-1, which was 207.5 (u/g.f.wt) The reason is that increased salt concentration causes plant stress, leading to an increase in reactive oxygen species (ROS) such as H2O2 and O2-. To resist these toxic compounds, the plant activates the antioxidant enzyme (POD) as a defensive mechanism; the enzyme decomposes H2O2, reduces oxidative damage within the cells, and protects cellular membranes from damage, thereby increasing the plant’s ability to tolerate salinity [18,19] and it agrees with the findings of [20] in his study on Solanum lycopersicum plants, However, in comparison to the control treatment, irrigation with high salinity levels increased peroxidase (POD) enzyme activity to twice its value.
The results in the mentioned table indicated that foliar spraying with nano-iron oxide significantly affected the leaf content of the peroxidase enzyme, as its content increased to (244.9, 297.0, 309.3)( u/g.f.wt) compared to the control treatment, which reached 205.6 (u/g.f.wt). This is attributed to the fact that nano-iron oxide possesses peroxidase-like activity and can catalyze oxidation reactions associated with hydrogen peroxide H2O2, indicating its ability to enhance peroxidase enzyme activity [21,22]. This is consistent with the findings of [23] on Artemisia scoparia, where the POD enzyme activity value reached 4.5( U/mg protein) at a concentration of 15 mg.L-1 of nano-iron oxide.
The leaf peroxidase enzyme concentration was significantly impacted by the combined interaction of the study parameters, irrigation water salinity and foliar spraying with nano-iron oxide. The F2S0 treatment had the lowest value, 100.4 (u/g.f.wt), whereas the F2S3 treatment had the greatest value, 598.0 (u/g.f.wt). The rationale is that nano-iron increased the activity of the superoxide dismutase (SOD) enzyme, enhanced plant development, and decreased oxidative stress [24]. This is consistent with [12] and is consistent with [25] in his study on Triticum aestivum, showing a significant effect at nan o-iron oxide concentrations of (200, 500 mg.L-1) and salinity levels of (7.5, 15 dS.m-1).

Ascorbate Peroxidase (APX) enzyme activity (U/g.f.wt)
Ascorbate peroxidase (APX) enzyme content (U/g.f.wt) was significantly impacted by irrigation water salinity levels, according to Table 5. The APX enzyme increased as salinity levels rose, reaching (38.6, 44.5, 51.8) u/g.f.wt at salinity levels of (4, 6, 8) dS.m-1, respectively, compared to the irrigation water level of 2 dS.m-1, which was 36.5 )u/g.f.wt(.The plant increases the activity of antioxidant enzymes like APX as a result of oxidative stress brought on by higher salt concentrations. As part of the plant’s defense mechanism, this enzyme is involved in the ascorbate-glutathione cycle, which deals with the buildup of hydrogen peroxide and free radicals [26]. This is consistent with the studies of [27]. Brassica napus L. study by inHe et al. in 2025 recorded a 35% increment in the APX enzyme activity at salinity level of 15 (dS. Nano-iron oxide foliar spraying increased leaf content of ascorbate peroxidase (APX) to (36.6, 48.5, and 51.2) u/g.f.wt in comparison with treatment control 36.1 u/g.f.wt, according to the results in Table 5 [28]. By treating the plant with nano-iron oxide, which improves APX enzyme activity, you can completely restore the balance between O2– and H2O2 in the plant and its photo-oxidative stress, thus helping maintain a green color. This role of solanimycin in relation to salt resistance for the plant remains to be established [29]. This is in line with the findings by [30] showed that nano-iron oxide in a concentration of 1 (mg. L-1).
The amount of the ascorbate peroxidase enzyme was affected by the two-way interaction of the irrigation water salinity and foliar spraying with nano-iron oxide in leaves. The highest value (63.2) u/g.f.wt for the F2S3 treatment while the lowest value (17.6) u/g.f.wt was found for the F1S1 treatment. The causal explanation is that the APX enzyme is elevated to a higher position as a protective mechanism to eliminate reactive oxygen species (ROS), especially hydrogen peroxide. This activity increased further when sprayed with nano-iron oxide [13]. These were consistant with the findings of [31] on the doses and have a significant effect with 1 mg in Phoenix dactylifera L. The highest increase in APX gene (OperonYR52587) was 3.28 U/g.f.wt at L-1 of nano-iron oxide.

CONCLUSION
The results obtained in this study emphasized the vital role of foliar spraying by iron oxide nanoparticles in the efficiency of the defense system of the chamomile plant in conditions of salt stress. NaCl-induced stress at different levels increases production of ROS and decreases the activity of the succus of the plant. On the contrary, iron oxide nanoparticles foliar spray increased antioxidant enzyme activities, especially APX, CAT and SOD activities, leading to oxidative damage reduction and restored cell membrane stability.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.