Mitigation of salinity stress in Mentha piperita L. through foliar application of chitosan–salicylic acid encapsulated ZnO nanoparticles

Abstract

Salinity stress harms plant growth and physiological characteristics. To investigate the effect of NaCl salinity stress (without salinity stress and 100 mM salinity) and foliar spray with: control (distilled water), zinc oxide nanoparticles (40 mgL^− 1), salicylic acid (1 mM), chitosan (10 mgL^− 1) and zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix (CS-Zn-SA NPs) (40 mgL^− 1) on growth and some physiological attributes of mint; a factorial experiment was conducted based on a completely randomized design. Independent effect of CS-Zn-SA NPs spraying increased plant dry weight, nitrogen, and potassium content, as well as essential oil yield. Moreover, 100 mM salinity stress reduced the mentioned traits. NaCl_{100 mM} × spraying with CS-Zn-SA NPs enhanced total phenolics and flavonoids content. The interaction effect of NaCl_{100 mM} × without foliar spraying enhanced proline, malondialdehyde, and sodium content. Treatment without salinity stress × foliar spraying of CS-Zn-SA NPs raised the magnesium, zinc, and iron content of mint. In general, it can be stated that the synergistic effects of combining salicylic acid, zinc, and chitosan partially enhanced the growth potential and improved physiological properties of peppermint plants under salt stress conditions. Following complementary field studies, the results would be advisable for the production section.

Introduction

Salinity stress is a major constraint in agricultural productivity, particularly in arid and semi-arid regions of the world. In medicinal plants, mild salinity stress may enhance the accumulation of secondary metabolites by stimulating metabolic pathways; however, severe salinity stress has detrimental effects on physiological and biochemical processes in plants^1–3. Excessive salt concentrations induce oxidative stress through the overproduction of reactive oxygen species (ROS), including superoxide radicals, hydrogen peroxide, singlet oxygen, and nitric oxide, which damage chloroplast structure, disrupt photosynthetic activity, and impair protein biosynthesis⁴. Other deleterious effects include reduced seed germination, inhibition of root and shoot growth, disturbed water relations, decreased osmotic potential, and ionic imbalance within the cell, ultimately resulting in osmotic stress and reduced cellular water potential^3–5.

One of the earliest consequences of salinity stress is water imbalance, as high salt concentrations in the soil reduce water potential, limiting water uptake by roots and inducing osmotic stress and physiological drought^1,4. Salinity also alters cell turgor thresholds, impairing nutrient absorption, photosynthetic efficiency, and overall plant growth^3,5. Ionic toxicity, primarily due to excessive Na⁺ and Cl^- accumulation, disrupts ion homeostasis and interferes with essential metabolic activities⁴. To mitigate these effects, plants activate complex antioxidant defense systems involving enzymatic and non-enzymatic components, as well as molecular strategies that regulate ion compartmentalization, gene expression, and hormonal signaling pathways such as those mediated by calcium and MAP kinase cascades⁶.

Salt stress causes significant physiological and biochemical alterations in Mentha species, directly affecting growth, yield, and the concentration of bioactive compounds. Increased Na⁺ and Cl^- levels in the rhizosphere lower soil osmotic potential, reducing water uptake and leading to decreased relative leaf water content, stomatal conductance, and chlorophyll concentration^7,8. Biochemically, elevated ROS production damages cellular macromolecules, while reductions in chlorophyll, K⁺/Na⁺ ratio, and soluble sugars further compromise photosynthesis and growth. Such changes result in decreased shoot biomass and increased root biomass, reflecting an adaptive strategy for stress tolerance^7,8.

In recent years, the application of nanoparticles and nanoformulations has emerged as an effective strategy to alleviate abiotic stress in plants, thereby improving agricultural sustainability under adverse conditions⁵. Nanoparticles enhance plant stress tolerance by mitigating oxidative damage, improving photosynthetic performance, and supporting nutrient utilization⁵. Among these, zinc (Zn) is an essential micronutrient involved in chlorophyll biosynthesis, photosynthesis, membrane stability, protein synthesis, and antioxidant defense⁹. Studies on rosemary (Rosmarinus officinalis)¹⁰ and Capsicum annuumL¹¹. have demonstrated that zinc nanoparticles alleviate salt-induced stress by reducing hydrogen peroxide and malondialdehyde levels and limiting the accumulation of Na⁺ and Cl^- ions.

Salicylic acid (SA), a phytohormone and signaling molecule, plays a crucial role in activating plant defense mechanisms under stress conditions. It mitigates oxidative stress by reducing ROS accumulation, enhancing photosynthesis, and increasing osmolyte content^1,12. In pea (Pisum sativum), SA pretreatment improved photosynthetic performance, elevated antioxidant enzyme activity, and upregulated Na⁺/H⁺ antiporter genes (SOS1, NHX1) under salinity stress¹. Increasing irrigation water salinity reduced physiological performance, yield, and postharvest quality of sour passion fruit, especially in the second cycle. Foliar application of salicylic acid (1.0–1.4 mM) in the first cycle partially alleviated salt stress and improved fruit yield and quality¹³.

Chitosan, a natural, biodegradable, and non-toxic biopolymer, exhibits antioxidant, antibacterial, and chelating properties¹⁴. It enhances plant tolerance to abiotic stresses by activating secondary signaling pathways¹⁵. Under saline conditions, chitosan improves photosynthesis, induces abscisic acid biosynthesis for stomatal regulation, and stimulates antioxidant defense through nitric oxide and hydrogen peroxide signaling, leading to precise osmotic adjustment and improved energy balance¹⁵. Exogenous application of chitosan and chitosan nanoparticles has been shown to alleviate salt-induced damage in Phaseolus vulgaris L. by improving pigment and carbohydrate content, enhancing antioxidant defense, and reducing oxidative stress markers¹⁴. However, excessive chitosan concentrations (≥ 2–3 gL^-¹) may exacerbate ROS production, impair photosynthesis, and disrupt hormonal homeostasis, thereby reducing plant growth^16–19.

Peppermint (Mentha piperita L.), an aromatic perennial herb of the Lamiaceae family, is widely utilized in the food, pharmaceutical, cosmetic, and perfumery industries. It is rich in bioactive constituents such as menthol, menthone, flavonoids, polyphenols, carotene, α-tocopherol, and betaine¹². Traditionally, peppermint has been used as an antipyretic, antispasmodic, antiasthmatic, antiemetic, antimicrobial, and anti-inflammatory agent, as well as for the treatment of liver disorders and rheumatism^20,21.

Like other medicinal plants, peppermint growth and productivity are adversely affected by abiotic stresses such as salinity. Therefore, evaluating the effectiveness of biostimulants that mitigate the impacts of salt stress is essential. The present study aimed to investigate the effects of foliar application of salicylic acid, chitosan, zinc nanoparticles, and zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix on the growth, physiological traits, and yield of Mentha piperita under salinity stress conditions.

Materials and methods

Plant material and experimental setup

The homogeneous mint (Mentha piperita L.) rhizomes were provided by a commercial cultivation site in Azar-Shahr County, Northwest Iran, in 2024, following the relevant institutional and national guidelines and legislations. The rhizomes were divided into uniform sections (approximately 15 cm in size) and surface sterilized with sodium hypochlorite (10% v/v)²² for 15 min, followed by washing with distilled water. Rhizomes were planted in 5-liter pots containing soil (Table 1). During the early establishment growth stage, plants were irrigated with unsalted water. Afterward, following 4 weeks, salinity treatments were imposed when plants were 10–13 cm (4–5 leaf stage). The salinity levels were 0 and 100 mM NaCl²⁰. Salinity began at 25 mM and then gradually increased to reach the final level within 12 days. Salinity treatment was based on the high salinity damage record in Northwest Iran and previous studies. To avoid salinity standing on the pot surfaces, the pots were regularly washed with tap water once a week. Foliar treatments were applied as sprays, and the solutions used included: dH₂O, nano ZnO (40 mg l^− 1)²³; salicylic acid (1 mM)¹²; chitosan (10 mgL^− 1)^24, and zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix (40 mgL^− 1). The nanoparticles used in this study were synthesized in the chemistry laboratory of Azarbaijan Shahid Madani University, Iran. Foliar applications were applied twice during the plant growing period. The first application was simultaneous with salinity exposure, and the second foliar spray was 2 weeks later. Two months after the second foliar treatment (at 10 weeks of salt stress), the plant samples were taken for further analysis. Every pot contained a single rhizome of 15 cm in length. Each experimental unit consisted of three pots, and the treatments had three replications. Irrigation of the plants was performed every 5 days using the specified salt treatment.

Table 1.

Physicochemical properties of the soil.

Soil texture	K content (mgKg−1)	P content (mgKg−1)	N content (%)	Organic matter (%)	Ca(CO3)2 (%)	EC (dSm−1)	pH
Sandy loam	150	6.1	0.13	0.6	1.2	1.5	6.5

Synthesis of chitosan-zinc-salicylic acid nanoparticles (CS-Zn-SA NPs)

Instrumentation

Fourier transform infrared (FTIR) analysis was done on a Vector 22 (Bruker, Ettlingen, Germany) Fourier transform infrared spectrometer using KBr as the mulling agent. The dynamic light scattering (DLS) measurements were taken on the Zetasiser instrument ZEN3600 (Malvern, UK MAL 1001767) with a He-Ne laser beam at 511 nm and 25^°C. An electronic analytical balance (PFB300-3, Kern, Germany) was used for weighing the solid materials.

Synthesis

The ionotropic gelation method was employed to synthesize chitosan-zinc-salicylic acid nanoparticles (CS–Zn–SA NPs). In this method, chitosan acts as a stabilizing and cross-linking biopolymer, while zinc ions and salicylic acid are incorporated into the chitosan network through ionic and hydrogen-bonding interactions. The resulting material is a hybrid nanocomposite in which zinc-containing domains are encapsulated within a chitosan–salicylic acid matrix, rather than ZnO nanoparticles merely surface-coated with chitosan–salicylic acid. Briefly, 0.15 g of chitosan (medium molecular weight, 75–85% deacetylated; Sigma-Aldrich) was dissolved in 100 mL of freshly prepared 1% (v/v) acetic acid under magnetic stirring (400 rpm) for 30 min until a clear solution was obtained. Zinc chloride (0.24 g) and salicylic acid (0.10 g) were then added sequentially under continuous stirring, and the pH of the solution was slowly adjusted to 9.0 by dropwise addition of 1 M NaOH to facilitate ionic interactions among chitosan, zinc, and salicylic acid. The mixture was stirred for 2 h at room temperature. Subsequently, 1 mL of 0.5% (w/v) sodium tripolyphosphate (TPP) solution was added dropwise to promote further cross-linking and nanoparticle formation, and stirring continued for 1 h until turbidity indicated nanoparticle formation. The suspension was centrifuged at 10,000 rpm for 15 min; the precipitate was washed twice with distilled water and dried at 70 °C for 4–5 h to yield CS–Zn–SA nanopowder. The purpose of this encapsulation strategy is to combine zinc as an essential micronutrient, salicylic acid as a signaling molecule, and chitosan as a biodegradable carrier and biostimulant for gradual release and synergistic action under salinity stress.

Similarly, chitosan-zinc nanoparticles (CS–Zn NPs) were synthesized using the same procedure, excluding the addition of salicylic acid to the chitosan solution²⁵. This allowed for an evaluation of salicylic acid’s influence on the efficiency and properties of the synthesized nanoparticles.

The fresh and dry weight of plants

The plant parts harvested at the early flowering stage (9 weeks after planting) were air-dried until constant weight. The fresh and dry weights were recorded by a digital scale (BB141, Beco, Germany).

Chlorophyll index

The chlorophyll index was determined by a portable chlorophyll meter (Erma, Japan).

The soluble solids content of the leaves

Soluble solid content (TSS) was quantified by a hand refractometer (Erma, Tokyo, Japan) from the extract obtained by squeezing the leaves, and the data are presented as ⁰Brix.

Relative leaf water content

First, the fresh weight of the leaf was determined (FW), then the leaf was floated in water in a closed container for 24 h, and its weight was measured again (TW). In the third step, the leaves were transferred to an oven at 70 °C for 24 h, and their dry weight was recorded (DW). The relative moisture content of the leaf was calculated using the following formula²⁶.

Ion leakage measurement

0.2 g of the leaf was placed in 10 ml of double-distilled water for four hours at 30 °C, and the electrical conductivity of the tissue was measured (EC1) after 3 h. Then, the samples were transferred to a hot water bath. The bath temperature was 100 °C, and the samples were kept at this temperature for 2 min. After cooling the samples, the electrical conductivity of the samples was measured with an EC meter (EC2)²⁷. The percentage of ionic leakage was calculated from the following equation:

Ionic leakage =(EC1/EC2) ×100.

Hydrogen peroxide content

Liquid nitrogen was used to grind 0.2 g of mint leaves. 5 mL of trichloroacetic acid (1% w/v) was added to the sample, and the resulting mixture was centrifuged at 12,000 × rpm for 10 min. 0.5 mL of the solution was mixed with 0.5 mL of 10 mM potassium phosphate buffer with pH 7.0 and 1 mL of potassium iodide. The absorbance of the samples was measured at 390 nm using a spectrophotometer²⁸.

Malondialdehyde content

0.5 g of mint leaves was ground in liquid nitrogen, and 5 ml of trichloroacetic acid (5% w/v) was added to the sample and centrifuged at 4000 rpm for 10 min. Two ml of the supernatant was mixed with 2 ml of 5% thiobarbituric acid + 20% trichloroacetic acid mixture, heated for 30 min at 95 °C, and then immediately placed on ice. The resulting mixture was centrifuged at 4000 rpm for 10 min, and the absorbance of the MDA was measured with a spectrophotometer (T80+, China) at 532 and 600 nm²⁹.

Proline content

0.5 g of leaf tissue was thoroughly homogenized with 10 mL of 3% sulfosalicylic acid in an ice-cold mortar. The resulting mixture was centrifuged at 10,000 rpm for 10 min. 2 mL of the obtained supernatant were mixed with 2 mL of ninhydrin reagent and 2 mL of glacial acetic acid. The tubes were placed in a boiling water bath at 100 °C for one hour and then cooled on ice. Subsequently, 4 mL of toluene was added to each tube, and the mixture was vigorously shaken to allow the formation of a colored phase. Proline content was measured using a spectrophotometer (T80⁺, China) at 520 nm².

Flavonoids and total phenolics content

10 ml of methanol was added to 5 g of dried mint leaves, and the samples were extracted for 12 h at room temperature. This procedure was repeated three times. After drying the methanol extract, methanol was added to the dried extract. 0.3 ml of aluminum chloride (10%) was added to 0.1 ml of the resulting methanol extract, and the mixture was kept at room temperature for 6 min. A spectrophotometer (T80, China) was used to measure the absorbance of the samples. The flavonoids absorbance was measured at 510 nm using rutin hydrate as a standard³⁰.

To measure the total phenolics content, the Folin-Ciocalteu reagent was used at 750 nm. Briefly, 1 mL of the methanolic extract was mixed with 0.2 mL of Folin–Ciocalteu reagent and 1 mL of 2% (w/v) sodium carbonate solution. The reaction mixture was allowed to stand at room temperature for 30 min, followed by centrifugation at 13,000 rpm for 5 min to remove particulates. The absorbance of the supernatant was measured at 750 nm using a UV–visible spectrophotometer (T80, PG Instruments Ltd., China). Gallic acid was used as the standard³¹.

Catalase activity

Catalase activity was determined spectrophotometrically by measuring the decrease in absorbance of hydrogen peroxide (H₂O₂) at 240 nm. Fresh mint leaves (0.2 g) were homogenized in ice-cold 0.1 M potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA. The homogenate was centrifuged at 12,000 × rpm for 10 min at 4 °C, and the supernatant was used as the enzyme extract. For the assay, 0.5 mL of enzyme extract was mixed with 1.5 mL of 0.1 M phosphate buffer (pH 7.0) and 1.45 mL of distilled water. The reaction was started by adding 0.5 mL of 75 mM H₂O₂, and the absorbance decrease at 240 nm was recorded for one minute. Catalase activity was expressed as µmol H₂O₂ decomposed per minute per milligram of protein (µmol H₂O₂ mg⁻¹ protein min⁻¹). Protein content was determined using the Bradford method. All steps were carried out at 4 °C to maintain enzyme stability³².

Superoxide dismutase activity

To measure superoxide dismutase activity, a reaction mixture was prepared in the dark by combining 50 mM potassium phosphate buffer (pH 7.5), 75 µM nitroblue tetrazolium (NBT), 13 µM methionine, 0.1 mM EDTA, and 4 µM riboflavin. To initiate the assay, 100 µL of the sample extract was added to 3 µL of the reaction mixture, and the reaction was started by exposing the mixture to a 40 W light source for uniform illumination. The absorbance of the samples was monitored at 560 nm at one-minute intervals for eight minutes³³. Appropriate controls were included, with the reaction mixture alone serving as a negative control to determine the maximum rate of NBT photoreduction, and a sample with known SOD activity as a positive control to validate the assay. The inhibition of NBT photoreduction in the presence of the enzyme indicated SOD activity, with greater inhibition corresponding to higher enzymatic activity. The assay was conducted at room temperature, and care was taken to maintain consistent light intensity and measurement conditions, as well as to keep the reaction within the linear range of absorbance for accurate quantification³⁴.

Total soluble proteins content

The total protein content of the leaves was measured by the Bradford method³⁵. 0.5 g of leaves were ground in 5 ml of tris-glycine buffer (pH 7.4) for 10 min. The supernatant was centrifuged at 12,000 rpm for 10 min at 4 °C to remove cellular debris. The resulting supernatant was collected as the crude protein extract. 100 µl of the supernatant was mixed with 5 ml of Bradford solution (Coomassie Brilliant Blue G-250), and after 10 min incubation at room temperature, the absorbance of the samples was read at 595 nm. Bovine serum albumin (BSA) was used as the standard for calibration, and the protein concentration of each sample was calculated from the standard curve and expressed as mg protein g^-1 fresh weight.

Elemental content

Healthy leaf samples were collected and thoroughly washed with deionized water, then dried at 65–70 °C and ground into a uniform powder. The powdered samples were digested with a mixture of nitric and perchloric acids (4:1) to completely decompose the organic matter. The resulting solutions were diluted with deionized water and analyzed for the content of mineral elements, including calcium, magnesium, iron, zinc, and copper, using an atomic absorption spectrophotometer (Shimadzu, AA6300, Japan). Na and K were measured with a flame photometer (Jenway, PFP7, Germany). Instrument calibration was performed with certified reference standards, and the accuracy and precision of the measurements were verified using control samples and quality checks^36,37.

Essential oil distillation and analysis

The harvested plants were hydro-distilled to determine the percentage (content) of essential oil using the Clevenger method of the British Pharmacopoeia for 3 h. To remove the water droplets, anhydrous sodium sulfate was added to each distilled essential oil, and then the oils were stored at 4 °C. The essential oil content and yield were calculated accordingly³⁸.

Experimental design and data analysis

The experiment was factorial based on CRD with three replications. Analysis of variance (ANOVA) was performed by MSTAT-C ver. 2.1. and SAS software version 9.4. Mean comparisons were done by the Least Significant Difference (LSD) test at 5% probability levels. Standard deviations were evaluated for the traits (n = 3). Pearson’s correlation and cluster dendrogram heat maps were depicted in R software (R Foundation for Statistical Computing, version 4.1.2).

Results

Characterization of synthesized CS-Zn-SA NPs

DLS technique measured the average hydrodynamic size of CS-Zn-SA NPs as 120 nm (Fig. 1).

Fig. 1.

Particle size distribution of CS-Zn-SA NPs.

The Fourier transform infrared (FTIR) spectroscopy can be used to determine the functional groups on the surface of CS-Zn-SA NPs, CS-Zn NPs, chitosan, and salicylic acid (Fig. 2A–D). The FTIR spectrum of chitosan displayed characteristic absorption bands at 3452 cm^-1 (O-H/N-H stretching), 2924 cm^-1 (C-H stretching), 1641 cm^-1 (C = O stretching in -CONH₂), 1383 cm^-1 (C-N stretching in -CONH₂), 1156 cm^-1 (C-O-C stretching), and 1092 cm^-1 (C-O stretching)^25,39 (Fig. 2A). The FT-IR spectrum of salicylic acid exhibited characteristic absorption bands at 3235 cm^-1 (O–H stretching), 2857 cm^-1 (C–H stretching), 1611 cm^-1 (C = O stretching), 1442–1483 cm^-1 (C–C stretching), and 1296 cm^-1 (C–O stretching) and 1247 cm^-1 (OH stretching), consistent with literature reports^25,40 (Fig. 2B). For CS-Zn NPs, (Fig. 3D) key bands appeared at 3424 cm^-1 (O-H), 2924/2856 cm^-1 (CH₃/CH₂), 1565 cm^-1 (C-H bending), 1262 cm^-1 (NH₂), 1022 cm^-1 (C-O), 827 cm^-1 (glucosidic bond), and 522 cm^-1 (Zn-O). The CS-Zn-SA NPs spectrum showed similar bands with slight shifts: 3423 cm^-1 (O-H), 2926 cm^-1 (CH₃), 1577 cm^-1 (C-C), 1240 cm^-1 (NH₂), 1155 cm^-1 (C-O), 829 cm^-1 (glucosidic bond), and 521 cm^-1 (Zn-O), along with a new salicylic acid band at ~ 1670 cm^-119. The peak shifts in CS-Zn-SA NPs (Fig. 2D) (chitosan/zinc bands to higher wavenumbers, salicylic acid bands to lower wavenumbers) confirm molecular interactions between all components while maintaining their structural integrity.

Fig. 2.

FT-IR spectrum of chitosan (A), FT-IR spectrum of salicylic acid (B), FT-IR spectrum of CS-Zn NPs (C), and FT-IR spectrum of CS-Zn-SA NPs (D).

Fig. 3.

Mean comparisons for the interaction effects of salinity and foliar spray on leaf length of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Aerial parts, fresh and dry weight, and leaf length

Plant fresh and dry weight was affected by the independent effect of salinity stress and foliar spray. Plant fresh (60.8%) and dry weight (73.4%) decreased with a salinity stress of 100 mM. The highest plant fresh and dry weight was observed in conditions without salinity stress (Table 2). Foliar sprays had a positive effect on plant fresh and dry weight, and CS-Zn-SA NPs improved fresh weight by 88% and dry weight by 78% compared to the control, respectively (Table 3). Moreover, leaf length was influenced by the interaction effect of experimental treatments. 100 mM salinity in conditions without foliar spray caused a significant reduction in leaf length. The use of foliar spray treatments had a positive impact on increasing leaf length in conditions of stress, and without salinity. The highest leaf length was observed in the treatment without salt stress × foliar application of CS-Zn-SA NPs (Fig. 3).

Table 2.

Mean comparisons for the effects of salinity on plant weight of Mentha piperita L.

NaCl (mM)	Aerial parts dry weight (g pot− 1)	Aerial parts fresh weight (g pot− 1)
0	4.9 ± 0.17a	46 ± 0.34a
100	1.3 ± 0.01b	18 ± 0.39b
Source of variation
Salinity (s)	**	**
Foliar spray (F)	*	**
S× F	ns	ns

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%.

Table 3.

Mean comparisons for the effects of foliar spray on plant weight of Mentha piperita L.

Foliar spray	Aerial parts dry weight (g pot− 1)	Aerial parts fresh weight (g pot− 1)
0	2.3 ± 0.12e	21.8 ± 1.2d
Nano ZnO	2.8 ± 0.11d	29 ± 1.3c
Chitosan	3.1 ± 0.84c	30 ± 0.98bc
Salicylic acid	3.4 ± 0.62b	31 ± 0.21b
CS-Zn-SA NPs	4.1 ± 0.21a	40 ± 0.28a

Similar letters show no meaningful difference at a 5% probability level (LSD test).

Chlorophyll index

Treatment without salinity stress enhanced the chlorophyll index (60% compared to 100 mM NaCl salinity) (Fig. 4A). Foliar spray improved the chlorophyll index. The highest chlorophyll index (41 SPAD) was observed in the foliar spray of CS-Zn-SA NPs, which shows a 70% increase compared to the treatment without foliar treatment. The lowest chlorophyll index was recorded in the non-foliar spray treatment (24 SPAD). In terms of chlorophyll index, no difference was observed between foliar spray with salicylic acid and chitosan and zinc nanoparticles + chitosan (Fig. 4B).

Fig. 4.

Mean comparisons for the effects of salinity (A) and foliar spray (B) on chlorophyll index of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Relative leaf water content

In non-stress conditions, salinity raised the relative leaf water content. With the intensification of salinity stress, the RWC decreased by 78% compared to the control (Fig. 5A). Foliar spray with nanoparticles improved the RWC compared to the control. In terms of RWC, no difference was observed between the foliar spray of salicylic acid and chitosan; both were at the same statistical level. The highest RWC was observed in the foliar spray of CS-Zn-SA NPs, which indicated a 24% increase compared to the control (Fig. 5B).

Fig. 5.

Mean comparisons for the effects of salinity (A) and foliar spray (B) on the relative water content of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Hydrogen peroxide and malondialdehyde content, and ion leakage percentage

The interaction effects of the experimental treatments affected hydrogen peroxide and malondialdehyde content, and the ion leakage. The foliar sprays under stress conditions had a positive effect on reducing the content of hydrogen peroxide, malondialdehyde, and ion leakage. In terms of ion leakage, no difference was observed between the nanoparticle treatments and the control under conditions without salt stress. Otherwise, the highest content of hydrogen peroxide, malondialdehyde, and the top ion leakage percentage ​​were observed under 100 mM salt stress without foliar spraying, which shows 88, 210, and 123% increase compared to control (no salinity and no foliar spray), respectively (Table 4).

Table 4.

Mean comparisons for the interaction effects of salinity and foliar spray on some physiological characteristics of Mentha piperita L.

NaCl (mM)	Foliar spray	H2O2 content (nmol mg− 1 FW)	Ion leakage (%)	Malondialdehyde (nmol mg− 1 FW)
0	0	98 ± 1.6d	17 ± 0.36 c	8.7 ± 0.02d
0	Nano Zn	118 ± 1.0b−d	16 ± 0.54c	6.5 ± 0.85de
0	Chitosan	101 ± 0.19d	12 ± 0.13c	4.7 ± 0.65e
0	Salicylic acid	114 ± 0.69cd	15 ± 0.10c	5.3 ± 0.25de
0	CS-Zn-SA NPs	105 ± 0.87d	12 ± 0.31c	4.7 ± 0.00e
100	0	184 ± 2.3a	38 ± 1.1a	27 ± 0.13a
100	nano Zn	138 ± 1.0b	30 ± 1.0b	18.3 ± 0.11b
100	Chitosan	135 ± 0.9 b	27 ± 0.11b	17.1 ± 0.21bc
100	Salicylic acid	125 ± 2.1bc	26 ± 0.21 b	14.4 ± 0.26c
100	CS-Zn-SA NPs	131 ± 1.9 bc	23 ± 0.98b	17.0 ± 0.24bc
Source of variation
Salinity (s)		**	**	**
Foliar spray (F)		**	**	**
S× F		**	*	**

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%. CS-Zn-SA NPs represent nano zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix.

Soluble solids content

The results showed that the highest soluble solids content was observed in treatments without salinity stress and 100 mM sodium chloride stress × spray of CS-Zn-SA NPs (Table 5).

Table 5.

Mean comparisons for the interaction effects of salinity and foliar spray on some nonenzymatic antioxidant content of Mentha piperita L.

NaCl (mM)	Foliar spray	Total soluble solids content (Brix°)	Proline content (µg g− 1 FW)	Flavonoids content (mgg− 1 FW)	Total phenolics content (mgg− 1 FW)
0	0	1.6 ± 0.23e	4.9 ± 0.21e	1.0 ± 0.00e	6.4 ± 0.14e
0	Nano Zn	2.2 ± 0.24cd	5.3 ± 0.02e	1.4 ± 0.23de	8.5 ± 0.27de
0	Chitosan	1.9 ± 0.13de	5.7 ± 0.21e	1.7 ± 0.02de	8.8 ± 0.46de
0	Salicylic acid	2.0 ± 0.14c−e	6.5 ± 0.25de	1.9 ± 0.21c−e	9.4 ± 0.08d
0	CS-Zn-SA NPs	3.1 ± 0.02a	5.8 ± 0.06e	1.3 ± 0.03cd	9.6 ± 0.71d
100	0	2.1 ± 0.17c−e	17.2 ± 1.06a	2.0 ± 0.01cd	8.0 ± 0.21de
100	nano Zn	2.4 ± 0.14b−d	15.8 ± 0.31b	1.3 ± 0.03cd	10.9 ± 0.31d
100	Chitosan	2.6 ± 0.28bc	11.8 ± 0.17c	2.6 ± 0.71c	13.5 ± 0.84c
100	Salicylic acid	2.5 ± 0.17bc	9.0 ± 0.84d	3.9 ± 0.21b	17.2 ± 0.09b
100	CS-Zn-SA NPs	2.8 ± 0.21ab	6.7 ± 0.21de	4.9 ± 0.36a	20.3 ± 1.51a
Source of variation
Salinity (s)		**	**	**	**
Foliar spray (F)		**	**	**	**
S× F		**	**	**	**

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%. CS-Zn-SA NPs represent nano zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix.

Proline content

The interaction effects of experimental treatments affected proline content of the plant. The conditions of without salt stress, without foliar spraying, and with foliar spraying of all compounds had the same effect on the proline content of the plant. Under 100 mM salinity × without foliar spraying, the proline content reached its maximum (17.2 µgg^− 1 FW), which shows up to 251% increase compared to the control. Foliar treatment under stress conditions decreased proline content (Table 5).

Phenolics and flavonoids content

The highest content of both compounds was observed under 100 mM stress conditions × foliar spraying of CS-Zn-SA NPs. In terms of phenolics content, no difference was observed between the treatments without salt stress in conditions without foliar spraying and foliar spraying of zinc nanoparticles and chitosan, and 100 mM salt stress in conditions without foliar spraying. The non-salt treatments × salicylic acid and CS-Zn-SA NPs, and 100 mM salt stress × zinc spray were the same in terms of their effects on phenolics content. For flavonoids content, salicylic acid × 100 mM salinity stress ranked second after spray with CS-Zn-SA NPs (Table 5).

Catalase and superoxide dismutase activity

The highest activity of both enzymes was observed at 100 mM salinity stress. In conditions without salt stress, the activity of both enzymes decreased (Table 6). All four foliar sprays increased the activity of superoxide dismutase, and the lowest activity of this enzyme was observed in the treatment without foliar spraying. Treatments without foliar spraying and spraying with zinc nanoparticles had the lowest catalase activity. Salicylic acid and CS-Zn-SA NPs treatments showed the highest catalase activity (Table 7).

Table 6.

Mean comparisons for the effects of salinity on catalase and superoxide dismutase activity of Mentha piperita L.

NaCl (mM)	Superoxide dismutase activity (units mg− 1 protein min− 1)	Catalase activity (µmol H2O2 mg− 1 protein min− 1)
0	96 ± 0.98 b	1.0 ± 0.36b
100	196 ± 3.1a	4.1 ± 0.21a
Source of variation
Salinity (s)	**	**
Foliar spray (F)	**	**
S× F	ns	ns

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%.

Table 7.

Mean comparisons for the effects of foliar spray on catalase and superoxide dismutase activity of Mentha piperita L.

Foliar spray	Superoxide dismutase activity (units mg− 1 protein min− 1)	Catalase activity (µmol H2O2 mg− 1 protein min− 1)
0	144 ± 2.6b	2.0 ± 0.27c
nano Zn	168 ± 0.87ab	2.2 ± 0.18 c
Chitosan	172 ± 1.41a	2.5 ± 0.17bc
Salicylic acid	176 ± 1.36a	2.7 ± 0.39ab
CS-Zn-SA NPs	192 ± 0.21a	3.1 ± 0.28a

Similar letters show no meaningful difference at a 5% probability level (LSD test).

Soluble proteins content

Salinity stress had an increasing effect on protein content. 100 mM salinity stress enhanced the soluble protein content by 26% compared to non-stressed conditions (Fig. 6A). Among the nanoparticles used, the application of CS-Zn-SA NPs raised protein content by 88% compared to the control (Fig. 6B).

Fig. 6.

Mean comparisons for the effects of salinity (A) and foliar spray (B) on protein content of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Nitrogen and phosphorus content

The independent effect of experimental treatments affected the nitrogen and phosphorus content of the plant. Stress caused a decrease in the nitrogen and phosphorus content of the plant, and the highest nitrogen content was observed in the no-saline stress conditions (41.7 mgg^− 1 DW). The lowest phosphorus content was recorded under 100 mM salinity stress (1.6 mgg^− 1 DW) (Table 8). The highest nitrogen and phosphorus content was obtained in the foliar treatment with CS-Zn-SA NPs (32.2 and 3.6 mgg^− 1 DW, respectively). The least nitrogen content was observed in the control (18 mgg^− 1 DW). In terms of nitrogen content, no difference was observed between the foliar spray treatments of zinc nanoparticles, chitosan, and salicylic acid (Table 9).

Table 8.

Mean comparisons for the effects of salinity on elemental content and K/Na ratio of Mentha piperita L.

NaCl (mM)	N content (mgg− 1 DW)	P content (mgg− 1 DW)	K content (mgg− 1 DW)	Ca content (mgg− 1 DW)	Cu content (mgg− 1 DW)	K/Na
0	41 ± 0.78a	4.7 ± 1.0a	55 ± 0.09a	17.1 ± 0.25a	3.7 ± 0.003a	26.7 ± 0.41a
100	15.9 ± 1.05b	1.6 ± 0.17b	31 ± 0.36b	4.9 ± 0.11b	1.5 ± 0.11b	1.3 ± 0.08b
Source of variation
Salinity (s)	**	**	**	**	**	**
Foliar spray (F)	**	**	**	**	**	**
S× F	ns	ns	ns	ns	ns	*

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *Significant difference at P ≤ 5%; **Significant difference at P ≤ 1%.

Table 9.

Mean comparisons for the effects of foliar spray on the elemental content of Mentha piperita L.

Foliar spray	N content (mgg− 1 DW)	P content (mgg− 1 DW)	K content (mgg− 1 DW)	K/Na ratio
0	18 ± 0.91c	1.4 ± 0.08c	35 ± 0.41d	1.7 ± 0.00 cd
nano Zn	28 ± 0.38b	1.47 ± 0.32c	42 ± 0.87c	1.5 ± 0.21d
Chitosan	28 ± 0.24b	2.7 ± 1.0b	49 ± 0.36b	2.0 ± 0.02bc
Salicylic acid	27 ± 0.00b	2.9 ± 0.26b	48 ± 0.41b	2.2 ± 0.04b
CS-Zn-SA NPs	32.3 ± 0.32a	3.6 ± 0.003 a	53 ± 0.21a	2.9 ± 0.21a

Similar letters show no meaningful difference at a 5% probability level (LSD).

Potassium and sodium content and K/Na ratio

The independent effect of experimental treatments influenced potassium content and the potassium-to-sodium ratio. The highest potassium content (55 mg g^− 1 DW) and potassium-to-sodium ratio were observed in the non-saline treatment (26.7%). By the salinity of 100 mM, potassium content was reduced to 31 mgg^− 1 DW (Table 8). The highest potassium content (53 mgg^− 1 DW) and potassium-to-sodium ratio were observed in the foliar spraying of CS-Zn-SA NPs. The lowest potassium-to-sodium ratio was observed in the treatment without foliar spray and foliar use of zinc nanoparticles (Table 9). The highest sodium content was observed under 100 mM salinity stress treatment in conditions without foliar spraying. The nanoparticles used in foliar spraying helped the plant survive under stress conditions by reducing the sodium content (Fig. 7).

Fig. 7.

Mean comparisons for the interaction effects of salinity and foliar spray on Na content of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Calcium and copper content

With a salinity of 100 mM, calcium content was reduced by 71% and copper by 59% compared to the control. The highest calcium and copper content was observed under no salinity (Table 8). The effect of foliar spray treatments on calcium content was differential. Treatments without foliar spray and spray with zinc nanoparticles did not affect calcium content. However, the use of chitosan and salicylic acid improved calcium content compared to the previous two treatments (Fig. 8A). Among the nanoparticles used, CS-Zn-SA NPs attained the highest copper content. The use of zinc nanoparticles had a similar effect to the control and did not enhance plant copper content. Otherwise, chitosan and salicylic acid treatment enhanced the copper content of plants (Fig. 8B).

Fig. 8.

Mean comparisons for the effects of foliar spray on Ca (A) and Cu (B) content of Mentha piperita L. Similar letters show no meaningful difference at a 5% probability level (LSD test).

Iron, zinc, and magnesium content

100 mM salinity stress in conditions without foliar spraying reduced the content of magnesium, zinc, and iron. Spraying of CS-Zn-SA NPs in conditions without salinity stress improved Fe (123%), Zn (211%), and Mg (211%) content compared to the control (Table 10).

Table 10.

Mean comparisons for the interaction effects of salinity and foliar spray on the elemental content of Mentha piperita L. plants.

NaCl (mM)	Foliar spray	Fe content (mgg− 1 DW)	Zn content (mgg− 1 DW)	Mg content (mgg− 1 DW)
0	0	13 ± 0.91ef	9.00.38de	1.7 ± 0.03e
0	nano Zn	13.5 ± 0.74ef	10.2 ± 0.21c−e	2.6 ± 0.00d
0	Chitosan	20 ± 1.03b	13.5 ± 0.74bc	3.4 ± 0.11c
0	Salicylic acid	19.6 ± 0.26b	16.4 ± 0.14b	4.1 ± 0.12b
0	CS-Zn-SA NPs	29 ± 0.98a	28 ± 0.34a	5.2 ± 0.39a
100	0	10 ± 0.17g	8.9 ± 0.85e	1.1 ± 0.02e
100	nano Zn	11.5 ± 0.014fg	11.8 ± 0.56c−e	1.5 ± 0.06e
100	Chitosan	14.5 ± 0.41de	11 ± 0.17c−e	2.7 ± 0.25 d
100	Salicylic acid	16.6 ± 0.31cd	12.8 ± 0.8b−d	2.6 ± 0.31d
100	CS-Zn-SA NPs	17.8 ± 0.25bc	16.2 ± 1.25b	2.7 ± 0.0d
Source of variation
Salinity (s)		**	**	**
Foliar spray (F)		**	**	**
S× F		**	**	**

Similar letters show no meaningful difference at a 5% probability level (LSD). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%.

Essential oil content and yield

The content and yield of essential oil were affected by the independent effect of experimental treatments. Salinity stress improved the content and yield of essential oil compared to the non-stressed conditions (Table 11). Based on the results, the foliar application of CS-Zn-SA NPs enhanced the content and yield of essential oil compared to the other foliar treatments. In terms of the content and yield of essential oil, no difference was observed between spraying with zinc nanoparticles, chitosan, and salicylic acid, and the lowest content and yield of essential oil were obtained in conditions without foliar spraying (Table 12).

Table 11.

Mean comparisons for the effects of salinity on essential oil content and yield of Mentha piperita L.

NaCl (mM)	Essential oil yield (kg ha− 1)	Essential oil content (%)
0	9.0 ± 0.36b	0.5 ± 0.02b
100	16 ± 0.032a	1.3 ± 0.08 a
Source of variation
Salinity (s)	**	**
Foliar spray (F)	**	**
S× F	ns	ns

Similar letters show no meaningful difference at a 5% probability level (LSD test). ns, non-significant; *significant difference at P ≤ 5%; **significant difference at P ≤ 1%.

Table 12.

Mean comparisons for the effects of foliar spray on essential oil content and yield of Mentha piperita L.

Foliar spray	Essential oil yield (kg ha− 1)	Essential oil content (%)
0	5.5 ± 0.1c	0.6 ± 0.03c
nano Zn	13.5 ± 0.4b	1.0 ± 0.10b
Chitosan	13.1 ± 0.03b	0.98 ± 0.08b
Salicylic acid	15.2 ± 0.15b	0.96 ± 0.14b
CS-Zn-SA NPs	27 ± 0.11a	1.4 ± 0.31a

Similar letters show no meaningful difference at a 5% probability level (LSD).

The heat map analysis according to the reaction of mint plants to salinity stress along with foliar applications of SA, ZnO-NPs, Cs, Cs-Zn-SA nanoparticles revealed that the traits including ion leakage, MDA, proline, oil yield and content, TSS, Na, H₂O₂, and antioxidant such as total phenolics and flavonoids content, and SOD and CAT activity had positive compliance to salinity stress. The other evaluated criteria, such as macro-and micronutrient content, K/Na ratio, aerial part dry and fresh weight, leaf length, SPAD, total soluble proteins content, and relative water content, had negative reactions to salinity stress and positive responses to Cs-Zn-SA nanoparticles under salinity stress. Also, three major classes of the assessed traits of plants were depicted by cluster analysis and dendrograms in this heat map under foliar applications of SA, ZnO-NPs, Cs, Cs-Zn-SA nanoparticles with and without salinity stress. Class I contained total phenolics and flavonoids content, SOD and CAT, TSS, oil content, and oil yield; class II contained ion leakage, MDA, proline, Na, and H₂O₂; and class III comprised N, K, Mg, P, Zn, Cu, Ca, Fe, K/Na ratio, aerial part dry and fresh weight, leaf length, SPAD, total soluble proteins content, and relative water content (Fig. 9).

Fig. 9.

The heat map (A) and the loading biplot (B) of the assessed physiological, biochemical, and growth traits of mint plants under salinity stress, along with the foliar application of SA, ZnO-NPs, Cs, Cs-Zn-SA nanoparticles. The heat map represents relative water content, malondialdehyde, H₂O₂ content, total soluble proteins, superoxide dismutase activity, catalase activity, proline, total soluble solids, ion leakage, oil yield, oil content, Na, K/Na, chlorophyll index, and nutrients content. T1, T2, T3, T4, T5, T6, T7, T8, T9, and T10 refer to control, ZnO-NPs, chitosan, salicylic acid, chitosan-Zn-salicylic acid, salinity stress (100 mM NaCl), ZnO-NPs + salinity stress, chitosan + salinity stress, salicylic acid + salinity stress, and chitosan-Zn-salicylic acid + salinity stress.

In general, heat map cluster analysis suggested five classes. Class I included the foliar application of Cs-Zn-SA nanoparticles without salinity stress. Class II comprised salinity stress at 100 mM NaCl salinity. Class III included control, SA, ZnO-NPs, and Cs foliar spraying. Class IV contained salinity stress with Cs-Zn-SA nanoparticles treatment, and finally, class V embraced salinity stress + SA, ZnO-NPs, and Cs foliar utilization (Fig. 9A).

The principal component analysis (PCA) and biplot analysis identified patterns in the relationships between variables and clusters under salinity with foliar applications, which comprised two major PCA. The two major PCAs included 89.1% of the total variance observed in the present research, indicating a large portion of the information in the dataset. The first and the second PCA explained 62.5% and 26.6% of the total variance, respectively. The evaluated properties, such as CAT, flavonoids, SOD, and total phenolics were grouped in the positive PCA direction, indicating that they are correlated with each other and contribute strongly to the first PCA. Finally, the results of the biplot determined three clusters that were observed in the heat map correlation, in which cluster 1 was linked to antioxidant and protective mechanisms in mint plants under salinity stress. Cluster 2 was correlated with growth and yield-related factors, such as oil content, oil yield, and nutrients. Cluster 3 indicated damage or stress markers, such as membrane damage (ion leakage and MDA) or physiological responses (proline content) (Fig. 9B).

Discussion

The superior performance of the zinc oxide nanoparticles encapsulated in a chitosan-salicylic acid matrix (CS-Zn-SA NPs) observed in our study likely arises from a triple synergistic interplay that distinctly outperforms the binary combinations formerly tested. Chitosan functions not merely as a passive carrier but as an active biocompatible matrix that: (i) enhances the stability and controlled release of ZnO NPs, (ii) improves foliar adhesion and cuticular penetration, and (iii) independently primes plant immunity^25,39,41,42. Concurrently, ZnO NPs provide bioavailable zinc essential for auxin metabolism and antioxidant enzyme function (SOD, CAT)^43–46, while salicylic acid potentiates systemic acquired resistance (SAR) and upregulates phenylalanine ammonia-lyase (PAL) activity^12,47,48. The encapsulation of SA within the chitosan matrix as opposed to free SA application likely protects SA from premature degradation and enables its sustained delivery, a critical advantage documented in recent nanoencapsulation studies. Our findings extend the current understanding of chitosan-SA nanocomposite applications. While Gohari et al.⁴⁹ demonstrated that CTS-SA NPs seed priming improved salinity tolerance in corn salad (Valerianella locusta), their formulation did not include a metallic nanoparticle component, and CTS-Mel NPs outperformed CTS-SA NPs in their system. In contrast, our tri-component CS-Zn-SA formulation achieved biomass increases (+ 88% FW, + 78% DW) substantially higher than those reported for CTS-SA alone, indicating that ZnO NP incorporation provides a non-redundant, complementary mechanism particularly through zinc-mediated stabilization of photosynthetic machinery and membrane integrity⁴⁶. This interpretation aligns with recent advances in nano-enabled stress tolerance. Enhanced antioxidant enzyme activity in chili pepper under salinity has been demonstrated using chitosan-selenium nanoparticles⁵⁰; yet, our CS-Zn-SA formulation exhibits superior efficacy, likely attributed to SA’s specific role in IAA biosynthesis and meristematic stimulation.Furthermore, studies on wheat demonastrated that treatments with chitosan-proline/glycine nanoparticles upregulate ion homeostasis genes (TaSOS1, TaNHX1) and polyamine metabolism genes (TaSAMDC, TaSPDS) in plant⁵¹. Although our study did not profile these specific transcripts, the pronounced improvements in osmotic adjustment and ion homeostasis observed are consistent with chitosan-mediated upregulation of such stress-responsive networks. Future transcriptomic studies should investigate whether CS-Zn-SA NPs similarly modulate SOS pathway genes⁵² and polyamine biosynthesis. Regarding the ZnO NP component, our results corroborate and extend those of Jalali et al.⁴³ in Dracocephalum moldavica and Hassani et al.⁵³ in Salvia leriifolia, both of which demonstrated that ZnO NPs mitigate salinity by enhancing antioxidant capacity and reducing MDA/H₂O₂ accumulation. However, our + 78% dry weight increase represents a marked improvement over the 20–35% increases typically reported for ZnO NPs alone, underscoring the emergent property of the tri-component nanoarchitecture. The chitosan-SA matrix likely addresses the limited translocation and rapid aggregation challenges that constrain ZnO NP efficacy in foliar applications. A critical insight emerging from this study is that not all chitosan-based nanocarriers perform equivalently. Our results demonstrate that incorporating ZnO NPs reverses this hierarchy, rendering CTS-Zn-SA NPs highly effective. This suggests that nanocarrier efficacy is formulation and species-dependent, and that multi-component systems can overcome the limitations of individual bioactives^22,25,39. The mechanistic basis whether ZnO NPs facilitate SA uptake, synergize with SA at the signaling level, or provide independent zinc nutrition warrants dedicated investigation. From a translational perspective, our findings address a critical gap identified in recent reviews: the need for multi-functional nanoformulations effective under realistic agricultural conditions. While the present study was conducted under controlled greenhouse conditions, the magnitude of biomass enhancement (+ 88%) suggests that CS-Zn-SA NPs could significantly improve medicinal plant productivity in salt-affected soils. Nevertheless, field validation remains essential, particularly regarding nanoparticle fate, soil microbiome interactions, and long-term environmental safety.

Salinity stress significantly reduced the chlorophyll index (SPAD value) in Mentha piperita, indicating impairment of the photosynthetic apparatus. Although SPAD measurements do not provide direct chlorophyll a and b contents, they are widely used as a reliable, non-destructive indicator of relative chlorophyll status and photosynthetic performance⁵⁴. The decline in chlorophyll index under salt stress is likely due to oxidative damage to chloroplast membranes and enhanced degradation of photosynthetic pigments caused by reactive oxygen species^1,6,14. Foliar application of chitosan–salicylic acid encapsulated ZnO nanoparticles improved the chlorophyll index, suggesting a protective role in maintaining chloroplast stability rather than directly increasing pigment biosynthesis. Zinc helps preserve membrane integrity and stabilize photosystem II^43,44, while chitosan^17,18 and salicylic acid^24,25 enhance antioxidant defenses and regulate stomatal behavior under stress. Nanoparticles may also improve nutrient use efficiency and photosynthetic performance, reflecting enhanced protection of the photosynthetic apparatus under salinity stress^17,24,44. Overall, the observed increase in SPAD values indicates improved chloroplast protection and stress tolerance rather than absolute chlorophyll content increase.

Under salinity stress, excessive ROS production overwhelms the plant’s defense system, leading to oxidative damage and cell death^50,55. The cell membrane is particularly vulnerable, and ion leakage serves as an early indicator of stress-induced damage, while malondialdehyde, a product of membrane lipid peroxidation, reflects oxidative stress^19,55,56. Studies have shown that NaCl-induced salinity increases ion leakage in Dracocephalum moldavica⁴³. Although low levels of H₂O₂ act as signaling molecules in metabolic processes and systemic resistance^45,46, excessive accumulation under stress is harmful. Application of chitosan under salinity conditions enhances antioxidant defenses, stabilizes cell membranes, and reduces MDA and H₂O₂ content in plants such as Catharanthus roseus⁴¹ and rice⁵⁷. These effects are attributed to the free radical scavenging activity of hydroxylated amino residues in chitosan^49,50,58. Additionally, chitosan improves carbohydrate and proline content, further strengthening the antioxidant system and enhancing yield under stress^41,50,58. Salicylic acid also mitigates stress by activating antioxidant enzymes (superoxide dismutase, peroxidase, and catalase), reducing ion leakage and lipid peroxidation, and promoting photosynthetic potential⁴⁷. In the present study, salinity increased H₂O₂, ion leakage, and MDA levels, confirming oxidative damage. Foliar application of CS-Zn-SA NPs effectively alleviated these effects, enhancing antioxidant defense and maintaining membrane integrity. This protective effect aligns with recent findings where Zn-SA-chitosan bionanoconjugates suppressed MDA and H₂O₂ accumulation in wheat under terminal heat stress by regulating antioxidant enzymes and cellular redox homeostasis⁵⁹. The superior efficacy of our tri-component formulation likely stems from the combined actions: chitosan provides structural stability and controlled release, ZnO NPs contribute to antioxidant enzyme function, and salicylic acid potentiates stress signaling pathways⁶⁰. This synergistic mechanism explains the comprehensive protection observed and supports the potential of CS-Zn-SA NPs as an effective strategy for salinity mitigation in medicinal plants.

Plants respond to salt stress by accumulating enzymatic antioxidants (catalase, superoxide dismutase, peroxidase) and non-enzymatic compounds (proline, sorbitol, menthol) to maintain osmotic balance, detoxify reactive oxygen species, and protect cellular structures and proteins^7,30,61. However, these intrinsic defense mechanisms are often insufficient, necessitating external intervention. Nanoparticles enhance plant stress tolerance by directly scavenging free radicals or stimulating antioxidant defense systems through signaling ions (e.g., calcium), signaling molecules (e.g., nitric oxide), plant hormones, and transcription factors^1,5,6,12,50. In this study, proline accumulation peaked under stress without foliar treatments, confirming its role as an osmoprotectant, whereas CS-Zn-SA NP application reduced proline content a clear indicator of stress alleviation. Similarly, soluble solids content was highest following CS-Zn-SA NP treatment, reflecting improved osmotic adjustment. These findings align with recent reports where chitosan-proline nanoparticles enhanced relative water content and reduced oxidative stress markers in wheat under salinity by upregulating ion homeostasis genes (TaSOS1, TaNHX1) and polyamine metabolism genes (TaSAMDC, TaSPDS)⁵¹. The reduction in proline content alongside improved osmotic balance suggests that CS-Zn-SA NPs mitigate stress perception rather than merely inducing tolerance mechanisms. Consistent with other studies, salinity induced oxidative stress, evidenced by increased peroxidase and polyphenol oxidase activities, enhanced glutathione levels, and improved membrane stability^{27,46,62,64,64}. Our study observed reduced plant biomass and physiological performance under salinity, highlighting the inhibitory effects of prolonged salt stress. Notably, CS-Zn-SA NP application improved antioxidant capacity and preserved cellular integrity, similar to responses in salt-tolerant mango cultivars⁶¹. These results corroborate a recent study on grapevines, where chitosan-salicylic acid nanocomposite (CS-SA NCs) at 0.5 mM alleviated salinity damage by reducing H₂O₂ and MDA while improving chlorophyll fluorescence parameters (Fv/Fm) and nutrient balance⁶⁵. Beyond ion regulation, zinc nanoparticles act as biochemical catalysts, enhancing antioxidant enzyme activities and modulating osmotic stress pathways. At the genetic level, nano-Zn regulates stress-responsive genes involved in aquaporin formation, ROS detoxification, and compatible solute synthesis^57,59,59. Recent transcriptomic analysis in tomato revealed that foliar ZnO NP application under salinity stress upregulates genes encoding antioxidant enzymes and stress-responsive transcription factors, while downregulating salt-induced senescence-associated genes⁶⁸. Nano-Zn also reduces membrane-bound NADH oxidase activity while boosting SOD, CAT, and peroxidase activities^66,67. As a key component of superoxide dismutase and Cu/Zn isomerase, zinc is critical for alleviating oxidative stress in plants. Chitosan complements these effects by inducing stomatal closure via abscisic acid signaling and activating antioxidant enzyme production through nitric oxide and hydrogen peroxide pathways⁶⁹. In white clover, chitosan nanoparticles increased simple sugars through upregulation of metabolic genes and enhanced sugar transport⁶⁹. The superior efficacy of our tri-component formulation likely stems from synergistic integration: chitosan provides controlled release and membrane stabilization, ZnO NPs supply bioavailable zinc for SOD activation and carbon metabolism, and salicylic acid potentiates systemic acquired resistance and phenylalanine ammonia-lyase activity^{64,67,69–71}. Salicylic acid enhances defense by increasing soluble sugars for osmotic adjustment, protein stability, and oxidative stress mitigation^58,69,72. It promotes phenolics and flavonoids accumulation, scavenging free radicals and protecting photosynthetic cells.

Application of SA and chitosan elev72 and Aconitum napellus⁵⁸. Proline accumulation under salinity stress activates antioxidant enzymes and helps reduce Na⁺/K⁺ ratio^6,61,73,74. Salinity stress impaired plant growth and disrupted mineral nutrition through excessive Na⁺ and Cl^- accumulation and reduced nitrogen content⁷⁴. The present findings closely agree with these reports: salinity (100 mM) declined essential nutrients while increasing sodium content. Proline accumulation supported ion homeostasis and antioxidant defense. Crucially, CS-Zn-SA NP application effectively mitigated salinity-induced nutritional imbalance by improving antioxidant activity and enhancing mineral uptake effects consistent with chitosan-silica nanoparticles loaded with salicylic acid that improved soil nutrient levels (N, P, K) and microbial community structure in ginseng cultivation⁷⁵. The multi-component CS-Zn-SA nanoformulation provides synergistic protection against salinity stress by: (i) chitosan-mediated membrane stabilization and controlled release, (ii) ZnO NP-driven antioxidant enzyme activation and nutritional fortification, and (iii) SA-potentiated stress signaling and secondary metabolism. This integrated mechanism explains the comprehensive improvements in biomass, osmotic adjustment, ion homeostasis, and antioxidant capacity observed in our study, positioning CS-Zn-SA NPs as a promising nano-biostimulant for sustainable crop production in salt-affected soils.

Under salinity stress, sodium uptake increases due to disturbances in cell wall integrity while potassium efflux via the GORK channel initially regulates membrane potential but subsequently enhances H⁺-ATPase activity and sodium accumulation^76–78. Retention of potassium and a high K⁺/Na⁺ ratio, mediated by KAT1, AKT1, and OsHKT2;1, is crucial for salt tolerance^78–80. Potassium also activates multiple enzymes, promoting protein biosynthesis and conserving energy for protective compound synthesis^78–80.

Chitosan enhances stress tolerance by activating salt-sensitive pathways, upregulating AsHKT1, and modulating biochemical processes such as callose deposition, cytosolic calcium, chromatin modification, and H⁺-ATPase inhibition^81,82. Foliar chitosan increases stress-responsive gene expression (P5CS, PIP, PAL), chlorophyll, proline, and nutrient content in Brassica napus⁸². Similarly, salicylic acid reduces sodium accumulation, improves nutrient uptake, photosynthesis, and antioxidant activity in Salvia officinalis⁶⁹.

Nano zinc acts as a molecular regulator of plant salt tolerance by enhancing potassium uptake, limiting sodium accumulation, and stabilizing cell membranes through upregulation of stress-related genes such as OsZFp, Gmzf3ol, and osISArn^46,83. It mitigates salinity by improving nutrient absorption, stimulating auxin production, and promoting root growth^52,64. Zinc application increases magnesium in rosemary¹⁰, elevates potassium in tomato²³, enhances K⁺/Na⁺ ratio in Dracocephalum moldavica⁴³, and improves phosphorus, calcium, and iron uptake in Ocimum basilicum⁶⁴.

The key finding of this study is that CS-Zn-SA NP application achieved superior ionic balance compared to individual treatments. This aligns with recent work showing that chitosan-silica nanoparticles loaded with salicylic acid improved N, P, and K availability in ginseng cultivation through enhanced nutrient mobilization⁷⁵. Similarly, chitosan-proline nanoparticles in wheat upregulated ion homeostasis genes (TaSOS1, TaNHX1) while reducing Na⁺ accumulation⁵¹. The synergistic effect observed here likely stems from: (i) chitosan-mediated regulation of ion transporters, (ii) ZnO NP provision of zinc for membrane stability and enzyme function, and (iii) SA-potentiated stress signaling that coordinates these responses. The co-application of proline or calcium with humic acid in salt-stressed pomegranate significantly reduced Na⁺ accumulation and increased K⁺, Ca²⁺, and K⁺/Na⁺ ratios⁸⁴. Our results extend these findings by demonstrating that a tri-component nanoformulation achieves comparable or superior outcomes through integrated mechanisms. Chitosan-mediated regulation of stress-responsive genes supports osmoprotectant accumulation, while SA and nano-zinc improve nutrient uptake and membrane stability. In conclusion, CS-Zn-SA NPs enhance salinity tolerance through coordinated regulation of ionic balance, osmotic adjustment, and stress-responsive pathways. This integrated response—consistent with recent nano-biostimulant research⁶⁵. allows plants to conserve energy and prioritize defense mechanisms, ultimately improving survival under salt stress.

Plant secondary metabolites are synthesized through biosynthetic pathways derived from primary metabolites. Terpenoids are produced via the mevalonate pathway in the cytosol or the methylerythritol-4-phosphate pathway in plastids^8,85. High salinity (150 mM) severely reduces essential oil biosynthesis in mint due to the toxic effects of sodium ions on cell membranes, cytoplasm, nucleus, and nutrient (NPK) uptake⁸⁶. However, studies on Pelargonium odoratissimum⁸⁷ and Mentha piperita⁸⁸ showed that salinity stress combined with zinc nanoparticle application increased essential oil content and enriched components such as geraniol, nerol, menthol, and menthone. Zinc enhances secondary metabolite biosynthesis by improving gene expression, antioxidant activity, enzyme function, and carbon metabolism under salt stress^46,87,88. Salicylic acid, a phenolic compound with hormonal activity, also contributes to secondary metabolite production and stress tolerance by activating multiple genes, altering primary metabolite levels, and reallocating carbon to secondary metabolite biosynthesis^58,71,89. Nevertheless, in Mentha piperita L., salicylic acid treatment alone did not significantly change essential oil composition³⁸. In the present study, 100 mM salinity increased both essential oil content and yield in peppermint. This finding aligns with the general stress response whereby plants enhance secondary metabolite biosynthesis as a protective mechanism^85,89. However, the key novel finding is that CS-Zn-SA NP application further amplified this increase beyond the level induced by salinity alone. This enhancement likely results from the synergistic effects of the tri-component formulation: chitosan activates sodium homeostasis (SOS1 pathway) and jasmonic acid signaling, ZnO NPs provide zinc essential for carbon metabolism and terpenoid biosynthesis, and SA facilitates carbon reallocation toward secondary metabolism^85,87. The combination of nutrient improvement and enhanced physiological efficiency consistent with previous studies explains the superior essential oil yield observed. Future research should focus on molecular and field-level investigations to better understand the mechanisms of CS-Zn-SA nanoparticle activity under salinity stress. In particular, exploring genetic and physiological tolerance responses across different plant species could provide valuable insights into species-specific nano-biostimulant efficacy.

Conclusions

The present study demonstrated that the application of CS-Zn-SA nanoparticles effectively mitigated the adverse effects of salinity stress on Mentha plants. The nanoparticles alleviated oxidative damage by enhancing both enzymatic and non-enzymatic antioxidant defense systems, as reflected by reduced malondialdehyde content and ion leakage. Foliar application of CS-Zn-SA NPs improved plant biomass, relative leaf water content, macronutrient (N, P, K, Ca) and micronutrient (Cu) content, total protein, and essential oil content and yield under saline conditions. Moreover, under 100 mM NaCl stress, treatment with CS-Zn-SA NPs significantly increased soluble solids, total phenolics, and flavonoids content, highlighting their potential in improving the quality and stress tolerance of mint plants. Otherwise, the current study was conducted under controlled greenhouse conditions, and field-scale validation is still required to confirm the reproducibility of these results under variable environmental conditions. Additionally, the long-term stability, environmental safety, and cost-effectiveness of nanoparticle applications should be further evaluated before large-scale agricultural adoption. Overall, the findings suggest that CS-Zn-SA nanoparticles represent a promising approach for enhancing salinity tolerance and productivity of mint plants, but further field-based and mechanistic studies are recommended to fully establish their practical applicability.

Abbreviations

CS-Zn–SA NPs

Zinc oxide nanoparticles encapsulated within a chitosan–salicylic acid matrix

CAT

Catalase

EC

Electrical conductivity

MDA

Malondialdehyde

RWC

Relative water content

SOD

Superoxide dismutase

TSS

Total soluble solids content

Author contributions

Conceptualization, L.V.M., and M.B.H.; data curation, M.H.R.A., M.H., and L.V.M. formal analysis and methodology, L.V.M., and F.R. project administration, L.V.M.; visualization, L.V.M., and M.B.H. writing original draft, L.V.M., and L.K. synthesis of nanoparticles, L.K.; writing—review and editing, L.V.M., and M.B.H. All authors have read and agreed to the published version of the manuscript.

Funding

The authors wish to thank the Azarbaijan Shahid Madani University, Iran, for the financial support of the study.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

All procedures were conducted following the relevant institutional, national, and international guidelines and legislation.