Enhanced salinity stress tolerance and growth promotion in Melissa officinalis using caffeic acid nanocomposites as a promising nano-bio-based strategy

Abstract

The application of caffeic acid (CA) and its nano-composite form (CA-NCs) has great promise for improving growth and abiotic stress tolerance because of the nano-composite formulation’s higher stability, bioavailability, and targeting. Limited research exists on caffeic acid nanocomposites in salinity-sensitive M. officinalis, with unexplored comparative efficacy against free caffeic acid in regulating photosynthesis, osmotic adjustment, and antioxidant activity. Thus, we assessed the potential of CA-NCs on growth and stress tolerance in Melissa officinalis grown during salinity compared to the free form of CA. The CA-NCs were synthesized and characterized through FTIR, TGA, and SEM analyses. CA-NCs demonstrated superior efficacy over free CA in mitigating NaCl stress in plants. Characterization confirmed their small size (99.8 nm), enhancing stability and bioavailability. Under salt stress, CA-NCs significantly improved root and shoot biomass, photosynthetic efficiency, and chlorophyll content. They effectively modulated osmotic stress by regulating relative water content and proline, while strongly reducing oxidative damage markers (MDA, H₂O₂). CA-NCs also boosted the activity of antioxidant enzymes and promoted the synthesis of protective secondary metabolites like flavonoids and anthocyanins. These synergistic effects highlight CA-NCs as a highly promising agricultural tool for enhancing plant growth and abiotic stress tolerance.

Introduction

Melissa officinalis L., a perennial herb from the Lamiaceae family, is commonly called lemon balm. This plant originates from the coastal areas of the Mediterranean and Western Asia. It is widely distributed in Iran, Europe, North America, South Africa, India, Ukraine, the Caucasus, and other regions [1]. Lemon balm is a bushy and upright plant, reaching a height of about one meter. Its leaves are soft, hairy, heart-shaped, and serrated, measuring 2 to 8 cm long. The flowers are complete and either white or pale pink. Traditionally, lemon balm has been used as a tonic, antispasmodic, anticancer, sedative, sleep aid, and memory enhancer. It is also used as a decoction, which exhibits antioxidant, antimicrobial, and antitumor properties [2]. Research indicates that lemon balm is relatively sensitive to salinity, tolerating up to 50 mM of salt; however, higher concentrations significantly reduce growth parameters such as stem height and relative water content while increasing the activity of defense enzymes [3].

Among the most destructive environmental stresses, salinity stress disrupts crop production on at least 20% of agricultural land worldwide [4, 5]. It is a significant threat to sustainable agriculture, causing disruptions in various physiological, biochemical, and molecular functions [6, 7]. Salinity stress disrupts germination, growth, photosynthesis, and stomatal conductance, reduces leaf water potential, increases reactive oxygen species (ROS), and disturbs ion homeostasis. This leads to impaired nutrient uptake and osmotic and ionic stress [8]. Plants mitigate these effects through various protective mechanisms, particularly ion balance, enhancing photosynthetic capacity, and improving antioxidant activity [9, 10].

Nanotechnology is a diverse field of knowledge that has rapidly developed in the past decades and found broad applications over the past couple of years [11, 12]. Nanomaterials, ranging between 1 and 100 NM, may now be used in various fields, such as agriculture [13]. This may open another way to conduct further studies on how nanomaterials may improve plants’ growth and raise their tolerance for environmental stress [14, 15]. Nanocomposites (NCs) are especially the subject of interest, which are nanomaterials possessing unique properties like high stability, small and very precise size, high solubility, and an appropriate surface area [16]. They can usually be divided into three main classes: metal, polymer, and ceramic matrix NCs. A wide variety of carriers are used to deliver materials to target cells and release them in a continuous and uniform manner for the preparation of nanocomposites. It includes polyvinyl alcohol, dextran sulfate, chitosan, cellulose, and its derivatives. Cellulose derivatives with polar groups, such as carboxymethyl cellulose, present great promise because they may act as solubilizers for poorly soluble substances, given the nature of their polar groups that include carboxyl methyl (CH₂COOH⁻) [17, 18].

Caffeic acid (CA) [3-(3,4-dihydroxyphenyl)prop-2-enoic acid] is a secondary plant metabolite that has many medicinal effects. It helps form some organic compounds related to plant stress tolerance, such as lignin [19, 20]. The stress-induced reactive oxygen species (ROS) lead to changes in physiological plant processes. These harmful ROS can damage organelles and membranes, leading to oxidation and disintegration of membrane structures [21]. Plants possess ROS counteracting systems consisting of enzymatic and non-enzymatic systems [22]. CA is an antioxidant that greatly contributes to plant physiology by restricting the activity of free radicals to prevent the oxidation of cell membranes and organelles. CA, however, has poor solubility and incompatibility profiles in standard solvent systems, restricting its use. Hence, the utilization of carriers is a significant factor in improving the efficiency of CA, which is central to this study [23, 24].

Herein, a carboxymethyl cellulose nanocomposite (CA-NC) was synthesized using CA and characterized in detail. The impact of this NC on lemon balm under salinity stress was subsequently assessed. Specifically, the study aimed to evaluate the defensive responses of the plant, including antioxidant mechanisms, levels of photosynthetic pigments, gas exchange parameters, and key secondary metabolites (tannins, anthocyanins, phenols, and flavonoids). The purpose of this research was to determine whether CA-NCs can provide stronger protective effects than free CA in enhancing Melissa officinalis tolerance to salinity stress. The role of these NCs in modulating plant defense could provide evidence of their effectiveness in improving plant resilience to environmental challenges, thereby offering promising possibilities for sustainable agricultural practice. We hypothesize that the nanoscale size and stability of CA-NCs enhance nutrient uptake and strengthen antioxidant defenses under salinity stress.

Materials and methods

Synthesis and characterization of NCs

CMC solution was prepared by dissolving CMC powder (0.4 mg/mL) in deionized water at 90 °C under constant stirring for 25 min. The resulting solution was then sonicated (40 kHz, 100 W) for 20 min to ensure complete dispersion. Separately, CA stock solution (2 mg/mL) was prepared in ethanol. The CA solution was added dropwise into the CMC solution at 65 °C using a syringe pump at a controlled rate of 1 mL/min under continuous stirring (300 rpm). During this process, the pH of the mixture was maintained at 6.5 ± 0.2. After complete addition, the mixture was ultrasonicated (40 kHz, 100 W) for 20 min to promote uniform nanocomposite formation, followed by centrifugation at 6000 rpm for 10 min. The resulting supernatant was carefully collected and poured into sterile Petri dishes for drying at 40 °C to obtain solid CA-NCs.

For characterization of the synthesized CA-NCs, FTIR spectra of CA, CMC, and CA-NCs were recorded in the range of 4000–400 cm⁻¹ (resolution 4 cm⁻¹) to identify functional group interactions. DLS analysis (Zetasizer Nano ZS, Malvern, UK) determined particle size distribution and polydispersity index (PDI) at 25 °C, with triplicate measurements. SEM (ZEISS, Germany, 15 kV) was used to examine morphology and surface features, with scale bars included for accuracy. TGA (TA Instruments, USA) evaluated thermal stability by heating samples (5 mg) from 25 to 700 °C at 10 °C/min under nitrogen.

Conditions for cultivation, treatments, and assessment of morphological parameters

Lemon balm seeds (Pakkan Seeds, Isfahan, Iran) were surface-disinfected with 1% NaClO, thoroughly washed, and germinated in autoclaved peat moss. At the 7-leaf stage, seedlings were transplanted into pots (20 cm diameter × 33 cm height) filled with 4 kg of a perlite–cocopeat mixture (50:50). Treatments (five replicates each) consisted of salinity (0, 50, 100, and 150 mM NaCl) applied alone [25] or in combination with CA or CA-NCs at 0.1 mg/mL as a soil drench. After 35 days of treatment, root length, shoot height, and fresh and dry biomass were measured (drying at 72 °C).

Photosynthetic parameters

Leaf tissue was homogenized in 80% acetone, centrifuged at 6000 rpm, and absorbance measured at 663, 645, and 470 nm for chlorophyll and carotenoids [26].

SPAD index was recorded using a CL-01 Chl meter. Fv/Fm was assessed with a PEA fluorimeter after 20 min dark adaptation. Photosynthesis rates were measured using a Walz IRGA (CO2: 500 mg L⁻¹, light: 800 µmol m⁻² s⁻¹).

Relative water content (RWC)

The RWC of lemon balm leaves was determined using the equation RWC (%) = [(FW-DW)/(SW-DW)] × 100, following the method of [27]. In this equation, FW represents the fresh weight of the leaf immediately after sampling, DW is the dry weight of the leaf, and SW is the saturated weight of the leaf after soaking in distilled water.

Proline content

For proline determination, fresh leaf samples were homogenized in 3% sulfosalicylic acid in an ice bath. After centrifugation, acetic acid and ninhydrin were mixed with the supernatants and incubated in a water bath (100 °C for 1 h). The samples were then transferred to an ice bath, and toluene was added. After vortexing for 20 s, the samples were kept at room temperature for a few minutes to allow the two phases to separate. The absorbance of the upper phase was then measured at 520 nm [28].

Oxidative stress markers

Fresh lemon balm leaves were homogenized with 1% trichloroacetic acid to measure hydrogen peroxide (H₂O₂) in an ice bath. The homogenized solution was then centrifuged at 12,000 rpm (15 min). After centrifugation, phosphate buffer and KI (1 M) were added to the supernatants, and the absorbance was recorded at 390 nm [29]. Lemon balm leaf samples were homogenized in 0.1% (W/V) trichloroacetic acid to measure malondialdehyde (MDA). The homogenate was centrifuged at 10,000 rpm for 10 min in a refrigerated centrifuge, and the supernatant was transferred to a new microtube. To the supernatant, 20% trichloroacetic acid containing 0.5% thiobarbituric acid was added and incubated at 95 °C for 15 min. After cooling mixtures with ice, their absorbance was monitored at 532 and 600 nm [30]. Electrolyte leakage (EL) from leaves was measured by recording the electrical conductivity of young lemon balm leaves after immersion in distilled water at 25 °C for 24 h (EC1) and at 100 °C in a water bath for 20 min, followed by cooling to 25 °C (EC2). The electrolyte leakage was calculated using the formula EC1/EC2 × 100 = EL [31].

Antioxidant enzyme assay

Leaves were homogenized in 0.01 M phosphate buffer (pH 6.8), centrifuged at 13,000 rpm (4 °C), and the supernatant was used for enzyme assays. Catalase (CAT) activity was measured per [32], ascorbate peroxidase (APX) per [33], and polyphenol oxidase (PPO) per Taranto et al. [34]. Protease activity was determined using 0.1% hydrolyzed casein, incubating at 45 °C for 1 h, stopping with 4% trichloroacetic acid, and measuring absorbance at 280 nm [35].

Total protein content

Fresh leaves were homogenized using liquid nitrogen, mixed with 0.1 M phosphate buffer, and centrifuged at 13,000 rpm for 20 min at 4 °C. The supernatants were mixed with Bradford reagent, and absorbance was measured at 595 nm. The protein concentration was then determined using a standard protein curve [36].

Total free amino acids and total soluble sugars (TSS)

Free amino acids were quantified by homogenizing leaves in 50 mM phosphate buffer (pH 6.8), treating with ninhydrin, incubating at 70 °C for 7 min, and measuring absorbance at 570 nm [37]. TSS was measured by homogenizing leaves in 80% ethanol, incubating at 95 °C for 60 min, mixing with anthrone reagent, and recording absorbance at 625 nm [38].

Phenolic compounds

Total flavonoids in the leaves were measured using a colorimetric method with AlCl₃. Plant samples were extracted with methanol, and the resulting extract was diluted with distilled water. Then, 5% NaNO₂ was added, followed by 10% AlCl₃ after 5 min. Finally, 1 M NaOH and distilled water were mixed with the solution and centrifuged at 4000 rpm (10 min). The absorbance of the supernatant was measured at 510 nm [39]. The methanolic extract was mixed with polyvinylpolypyrrolidone and incubated at 4 °C for 15 min to measure tannins. The mixture was then centrifuged at 3000 rpm for 15 min at 4 °C, and the absorbance of the supernatant was measured at 760 nm. The difference in absorbance between the raw and tannin-free extracts indicates the tannin content [40]. Leaf samples were homogenized in 96% ethanol to determine total phenols and left in the dark for 24 h. Ethanol (95%), Folin-Ciocalteu reagent (50%), and sodium carbonate (5%) were then added to the resulting solution. After keeping the mixture in the dark (1 h), the absorbance was measured at 725 nm. The concentration was determined using a standard curve [41]. Fresh leaves were ground with acidic methanol (1% HCl) to determine anthocyanins. The extract was left in the dark at 25 °C for 24 h. The mixture was then centrifuged at 4000 rpm for 10 min at 4 °C, and the absorbance of the supernatant was measured at 550 nm [42].

Statistical analysis

The data were analyzed using SPSS software (ver. 23). Tukey’s post-hoc test was used for group comparisons. Results are presented as mean ± standard devision. A p-value of ≤ 0.05 was considered statistically significant.

Results

Features of the synthesized CA-NCs

The FTIR spectrum for CA and CA-NCs, as shown in Fig. 1A and B, indicates that the wavenumber corresponding to the carbonyl group of CA has decreased from 1645 cm⁻¹ to 1635 cm⁻¹ in CA-NCs. This shift suggests the formation of hydrogen bonds between the carbonyl group of CA and the hydroxyl groups of CMC; as a result of this interaction, the frequency and bond energy decrease while the bond length within the carbonyl group increases. This frequency shift indicates the formation of a compound between CMC and CA. The particle shape and size of the NCs were evaluated using SEM and DLS. The average size of the CA-NCs particles is 99.8 ± 7.7 nm, with most particles being 99.4 nm. The hydrodynamic radius is 3301 nm (Fig. 1C). In the SEM analysis, the morphology of the particles ranged from amorphous to spherical (Fig. 2A).

Fig. 1.

Fourier-transform infrared (FTIR) spectra of (A) CA and (B) CA-NCs confirming the characteristic functional groups and interactions. C Particle size distribution of CA-NCs as analyzed by DLS, showing average hydrodynamic diameter and frequency distribution

Fig. 2.

Morphological and thermal characterization of CA-NCs. A SEM micrograph of CA-NCs at high magnification with size indications (scale bar = 1 μm). B Thermogravimetric analysis (TGA) curve of CMC. C TGA curve of CA. D TGA curve of CA-NCs, indicating thermal decomposition steps and mass loss percentages

The TGA curves for CA, CMC, and the synthesized Ca-NCs provide valuable insights into the thermal stability and composition of the materials. The higher residual mass of the NCs compared to pure CA indicates that incorporating CMC enhances the composite’s thermal stability. This is likely due to the strong interaction between CA and the hydroxyl groups of CMC, as suggested by the FTIR analysis. The reduction in mass loss of the NCs relative to CMC also demonstrates that the formation of the NCs reduces the overall thermal degradation of CMC, likely because the hydrogen bonding between CA and CMC restricts the mobility of polymer chains, thereby enhancing stability. Furthermore, the TGA curves exhibit multiple degradation steps. The first weight loss, observed at lower temperatures, can be attributed to the evaporation of water and other volatile components. The second stage corresponds to the degradation of organic components, such as CA and CMC. The delayed onset of this degradation in the NCs compared to pure CA suggests improved thermal resistance, which confirms the formation of a stable nanostructure through the interaction of CA and CMC (Fig. 2B, C, D).

Growth and physiology

The analysis of the results showed that the growth parameters of lemon balm under salinity stress conditions significantly decreased. The observed reductions at 50, 100, and 150 mM included decreases of 46.1%, 57.6%, and 83.5% in fresh root weight; 2.2%, 20.1%, and 67.6% in dry root weight; 32.5%, 40.8%, and 74.3% in fresh shoot weight; 33.2%, 43.2%, and 61.7% in root length; and 31%, 34.5%, and 41.7% in shoot length, respectively. CA and CA-NCs significantly improved the growth parameters at various salinity concentrations. The observed increases were 70.1%, 46.7%, and 104.3% in fresh root weight; 12%, 37.5%, and 67.6% in dry root weight; 45%, 51.5%, and 168.6% in fresh shoot weight; 58.6%, 51.4%, and 122.8% in root length; and 62.1%, 81.8%, and 53.1% in shoot length under CA treatment. Under CA-NCs treatment, the increases were 59.3%, 21.1%, and 64.1% in fresh root weight; 16.1%, 13.9%, and 28.6% in dry root weight; 33.1%, 38.1%, and 120.6% in fresh shoot weight; 4.2%, 113.3%, and 138.3% in root length; and 34.5%, 47.3%, and 22.5% in shoot length, respectively. The observed reduction in dry shoot weight at salinity concentrations of 100 and 150 mM was significant, amounting to 30.6% and 69.7%. At the same time, treatment with CA and its NCs increased dry shoot weight by 46.8% and 171.55% by CA and 74.8% and 155.04% by NCs, respectively (Table 1).

Table 1.

The effect of caffeic acid (CA) and caffeic acid nanocomposite (CA-NCs) on the morphological traits of Melissa officinalis (lemon balm) under salt stress (S, 50, 100, and 150 mM)

Treatments	Root length (cm)	Shoot length (cm)	Root fresh weight (g)	Root dry weight (g)	Shoot fresh weight (g)	Shoot dry weight (g)
Control CA CA-NCs	50.3 ± 7.35ef 60.0 ± 2.88f 75.0 ± 8.60g	28.0 ± 1.15def 31.3 ± 2.96ef 32.0 ± 4.16ef	11.16 ± 0.07j 14.11 ± 0.02k 09.71 ± 0.14h	3.24 ± 0.03f 6.55 ± 0.02j 4.74 ± 0.09i	12.63 ± 0.18f 12.34 ± 0.38f 11.51 ± 0.21e	3.60 ± 0.93e 6.14 ± 0.16g 4.45 ± 0.16f
S50 S50-CA S50-CANCs	33.6 ± 1.85bc 53.3 ± 1.52ef 35.0 ± 1.52bcd	19.3 ± 0.88abc 31.3 ± 2.33ef 26.0 ± 2.08cdef	06.02 ± 0.04f 10.24 ± 0.02i 09.59 ± 0.05h	3.17 ± 0.04f 3.55 ± 0.03g 3.68 ± 0.02h	08.52 ± 0.10c 12.35 ± 0.14f 11.34 ± 0.12e	3.60 ± 0.10e 3.15 ± 0.01d 2.86 ± 0.03c
S100 S100-CA S100-CANCs	28.6 ± 2.02ab 43.3 ± 2.40cde 61.0 ± 1.15f	18.3 ± 0.88ab 33.3 ± 2.02f 27.0 ± 2.64cdef	4.73 ± 0.04d 6.94 ± 0.02g 5.72 ± 0.01e	2.59 ± 0.03d 3.56 ± 0.02g 2.95 ± 0.02e	07.48 ± 0.21b 11.33 ± 0.06e 10.33 ± 0.06d	2.50 ± 0.54b 3.67 ± 0.08e 4.37 ± 0.03f
S150 S150-CA S150-CANCs	19.3 ± 0.88a 43.0 ± 1.73cde 46.6 ± 3.52de	16.3 ± 0.88a 25.0 ± 3.21bcde 20.3 ± 2.60abcd	1.84 ± 0.04a 3.76 ± 0.01c 3.02 ± 0.03b	1.05 ± 0.02a 1.76 ± 0.02c 1.35 ± 0.02b	3.25 ± 0.07a 8.73 ± 0.37c 7.17 ± 0.04b	1.09 ± 0.01a 2.96 ± 0.79 2.78 ± 0.04c

Means (± SD, n = 6) followed by the same letter are not significantly different (Duncan test; P < 0.05)

Salinity at 100 and 150 mM declined carotenoids by 18.3% and 45.2%, chlorophyll b by 26.4% and 48.8%, and chlorophyll a by 34% and 53.7%, respectively, over controls. In contrast, at both these salinity levels, CA and CA-NCs significantly increased the levels of all three photosynthetic pigments, with the NCs form of CA inducing a more pronounced enhancement (Fig. 3A, B, C). Although the two higher salinity levels significantly reduced Fv/Fm levels, adding CA and CA-NCs restored them (Fig. 3D). Both SPAD and stomatal conductance significantly decreased over the control at all levels of NaCl, with the lowest values of both traits observed at the highest NaCl level. In contrast, CA and its NC form significantly improved both traits in NaCl-stressed plants, with the improvement greater in the plants treated with CA-NCs (Fig. 3E, F).

Fig. 3.

Effects of caffeic acid (CA, 0.1 mg/mL) and caffeic acid nanocomposites (CA-NCs, 0.1 mg/mL) on photosynthetic traits of Melissa officinalis under salt stress (0, 50, 100, and 150 mM NaCl). A Chlorophyll a, B Chlorophyll b, C Carotenoids, D SPAD value, E Fv/Fm, and (F) Stomatal conductance. Data are means ± SD (n = 6). Columns with the same letter are not significantly different according to Duncan’s test (P < 0.05)

Salinity application reduced RWC and augmented leaf proline content, with the most significant changes observed under 150 mM salinity stress, showing a 46% decrease in RWC and a 54% increase in proline content. In contrast, applying CA and its NC form at all three salinity levels increased both parameters (Fig. 4A, B). 50, 100, and 150 mM NaCl increased TSS accumulation by 22.9%, 74.3%, and 76.2%, respectively, over the control. However, applying the CA-NCs under 100 mM NaCl and CA and CA-NCs under 150 mM NaCl induced significant increases in TTS over the corresponding salinity stresses alone (Fig. 4C).

Fig. 4.

The effect of caffeic acid (CA, 0.1 mg/mL) and caffeic acid nanocomposite (CA-NCs, mg/mL) on relative water content (RWC, A) and contents of proline (B), soluble sugars (C), and protein (D) of Melissa officinalis under salt stress (S, 50, 100, and 150 mM). Data are means ± SD (n = 6). Columns with the same letter are not significantly different according to Duncan’s test (P < 0.05)

Salinity stress led to a decreasing trend in total protein content and an increasing trend in total amino acids, with the most significant changes observed under 150 mM salinity. However, applying CA and its NCs increased both parameters at all salinity levels. The CA-NCs exhibited greater inductive effects than the CA form (Figs. 4D and 5A).

Fig. 5.

The effect of caffeic acid (CA, 0.1 mg/mL) and caffeic acid nanocomposite (CA-NCs, mg/mL) on contents of total amino acids (A), hydrogen peroxide (H₂O₂, B), malondialdehyde (MDA, C), electrolyte leakage (EL, D) and activity of catalase (CAT, E) and ascorbate peroxidase (APX, F) of Melissa officinalis under salt stress (S, 50, 100, and 150 mM)

Defense and oxidative stress

Over the control, 100 and 150 mM NaCl significantly increased H₂O₂ and MDA contents, with the highest accumulation observed under the 150 mM level. In contrast, CA and CA-NCs at both salinity levels reduced the accumulation of these compounds (Fig. 5B, C). The increase in NaCl stress led to a rise in EL, with the highest increase (95%) over the control at the 150 mM level. Both forms of CA significantly reduced leaf EL at all three salinity levels, with CA-NCs demonstrating a greater reduction effect (Fig. 5D).

The activity of CAT and APX showed an increasing trend with the rise in salt stress in the leaves. The highest activity of these enzymes under salt stress alone was observed in the 150 mM treatment. Additionally, the addition of CA and CA-NCs to NaCl treatments resulted in a further increase in the activity of these enzymes, with the CA-NCs treatment leading to a greater elevation in enzyme activity than CA treatments (Fig. 5E, F).

Salinity treatments alone or combined with CA or CA-NCs caused a significant rise in leaf protease activity. However, at all salinity levels, this enzyme activity’s highest and lowest levels were associated with salinity treatment alone and NaCl + CA-NCs, respectively (Fig. 6A). Adding NaCl treatments alone or in combination with CA or CA-NCs stimulated the activity of leaf PPO. However, except for the 50 mM salt level, where the NaCl + CA or CA-NCs treatments showed a significantly higher increase than NaCl alone, no significant differences were observed between treatments at the other two salt levels (Fig. 6B).

Fig. 6.

The effect of caffeic acid (CA, 0.1 mg/mL) and caffeic acid nanocomposite (CA-NCs, mg/mL) on the activity of protease (A) and polyphenol oxidase (PPO, B) and the content of total flavonoid (C), total phenol (D), total tannin (E), and anthocyanin (F) of Melissa officinalis under salt stress (S, 50, 100, and 150 mM)

Secondary metabolites

The total flavonoid content increased significantly under NaCl treatments, with the greatest increase observed in the 150 mM level. When combined with CA or CA-NCs, this increase was further enhanced. Notably, the CA-NCs treatment yielded the highest total flavonoid content at 150 mM NaCl (Fig. 6C). At all NaCl concentrations, adding CA or CA-NCs resulted in higher phenol levels than salt alone. The greatest increase was observed in the CA-NCs treatment at 150 mM NaCl, suggesting a synergistic effect of the CA-NCs and salt stress (Fig. 6D). Salt stress alone increased total tannin content, but adding CA or CA-NCs further amplified this effect. At 50 mM NaCl, the NaCl + CA or CA-NCs treatments showed significant increases in total tannin. In 100 and 150 mM, no significant difference was found between the treatments, but overall, adding CA or CA-NCs promoted an increase in tannin content (Fig. 6E). The total anthocyanin content was significantly enhanced by salt stress and the addition of CA or CA-NCs. At 50 mM NaCl, the combination of NaCl and CA-NCs led to the highest anthocyanin content. At higher NaCl concentrations, the CA-NCs treatment consistently resulted in the highest anthocyanin levels, indicating that the CA-NCs treatment, especially under higher salinity, led to a pronounced increase in anthocyanin production (Fig. 6F).

Discussion

The synthesized CA-NCs exhibited significant improvements in stability and bioactivity compared to pure CA, as indicated by the FTIR, TGA, and SEM analyses. The FTIR spectrum showed a shift in the carbonyl group of CA, suggesting successful interactions with CMC, which likely enhances the bioavailability and stability of the composite. These interactions are consistent with studies by [17], who observed similar hydrogen bonding between quercetin and biopolymers to improve stability and functionality in customized NCs. The particle size analysis revealed an average size of approximately 99.8 nm, which is in the optimal range for nanomaterials to enhance surface area and facilitate better uptake by plant cells, similar to findings by [43], who reported improved efficacy of nanoparticle-based treatments in plants. Furthermore, the TGA analysis proved that the CA-NCs presented better thermal stability than the CA form, probably due to the good interaction between CMC and CA. Moreover, this will make NCs more suitable for agricultural purposes since the environmental conditions are highly variable. These data generally evidence that CA-NCs showed better stability, bioavailability, and activity than free CA. All the data proved our hypothesis that the NCs are more efficient for alleviating plant stress, especially under conditions like salinity.

The current study of CA and its NCs showed the capability of improving growth attributes in M. officinalis plants under saline conditions. As expected, salinity stress drastically reduced growth and development, including fresh and dry root and shoot weights and root and shoot length. Results were in tune with previous ones, indicating that growth inhibition during salinity is attributed to osmotic stress and ion toxicity under salinity in different plant species [44]. However, treatment with CA and CA-NCs considerably mitigated the harmful impacts of salinity on growth traits. Apparently, the more effective role of the nanoformulation of CA on fresh shoot weight and root length was obtained in the case of the highest salinity level used (150 mM), where CA-NCs increased the two previous growth traits up to 168.6% and 138.3%, respectively. This agrees with the study documented by Haydar et al. [45], which mentioned that nanocarriers can cause plant growth improvement via improved nutrient and water uptake, but also by being a more efficient way of delivering bioactive agents. In this connection, the enhanced efficiency of CA-NCs was attributed to the nanoscale of the particle size, about 99.8 nm, enabling better permeability through plant tissues, hence the elevation in the bioavailability of CA. Considering photosynthetic performance, salinity stress severely declined pigment contents, known as sensitive indicators of oxidative stress in plants [9, 46]. Treatments with CA and CA-NCs recovered these pigments, while more recovery was observed in the case of CA-NC treatments. This suggests that the NC form of CA is better at alleviating oxidative damage, probably due to better cell internalization and sustainable release of CA, which acts as an antioxidant molecule [47]. In this respect, Arshneshin et al. [17] reported the same improvements in the pigment content of plants treated with NCs of phenolic compounds under salinity. Both CA and CA-NCs treatments significantly enhanced the Fv/Fm ratio and SPAD value under salinity stress, hence recovering photosynthetic efficiency and general plant vigor. Such increased efficiency of CA-loaded NCs in enhancing stomatal conductance and SPAD values, compared to free CA, draws support from work carried out by Haydar et al. [45], in which the authors showed that NCs improve gas exchange due to improved stomatal regulation. This rise in stomatal conductance may provide ways for improved CO₂ assimilation and WUE, which are considered very important for plant growth under abiotic stresses.

Salinity leads to osmotic imbalance in plants, reducing RWC and increasing the accumulation of osmolytes like proline, which helps cells maintain their water potential and prevent damage due to oxidative stress. The observed significant decrease in RWC and increase in proline at 150 mM NaCl are in line with the findings of Rhaman et al. [48], who established that salinity-induced osmotic stress often triggers the synthesis of proline as an adaptive response to safeguard cellular structures and proteins. Proline stabilizes cellular membranes and proteins by acting as a molecular chaperone, preventing the denaturation of enzymes and proteins under stress [49]. Interestingly, the application of CA and its NC form improved both RWC and proline content under salinity, with CA-NCs showing a stronger effect than free CA. This might suggest that the NC form enhances the plant’s ability to retain water and produce proline due to better uptake and more efficient cellular integration of the active compounds. This mechanism is well-documented in studies such as [17], which proposed that NCs, by enhancing the bioavailability of bioactive compounds, would more effectively modulate osmotic stress responses. Along with proline, TSS plays a crucial role in osmotic regulation through stabilizing proteins and cell membranes under stress. Salinity-induced increases in TSS suggest that plants use these sugars to cope with osmotic pressure [9]. TSS acts as an osmoprotectant, helping to balance water potential between cells and extracellular spaces [50, 51]. CA and CA-NCs raised the accumulation of TSS at 100 and 150 mM NaCl in this paper. The higher increase in the content of TSS during treatment with NCs indicates their ability to more effectively control osmotic balance. Such an improvement has most probably been caused by the sustained release and higher bioavailability of CA, which affects the biosynthesis of osmoprotective molecules like sugars. Shahmarbiglou and Razavi [52] and Arshneshin et al. [17] have reported similar changes in the increase of TSS levels under treatment with NCs of phenolic compounds; this results in an increased osmotic adjustment.

Here, salinity stress also reduced the total protein content and increased the total amino acids, which is quite a common phenomenon under stress conditions, whereby plants degrade proteins to synthesize amino acids for protection and metabolic functions [53]. Increasing amino acids is a very important function, especially when activating antioxidant enzymes and supporting cellular functions against stress. Salinity-induced reductions in protein and amino acid levels were reversed upon treatment with CA and CA-NCs, with the latter being more effective. This may indicate the possibility of enhancements in the synthesis of protein and amino acids by the NC form of CA through enhancing nutrient and water uptake, reducing oxidative injury, and facilitating the routes of protein synthesis pathways [54, 55]. These findings are in concert with previous reports by Shahmarbiglou and Razavi [52] and Sepehry Javan et al. [43], showing improved protein synthesis and minimization of oxidative injury as evidence for the positive impacts of phenolic-based NCs on enhancing the plant’s tolerance to stresses.

During salinity, obvious oxidative damage was present, reflected by the higher values of H₂O₂, MDA, and EL. Treatments with CA and CA-NCs exhibited a remarkable ability to reduce such effects under the salinity treatment, and NCs were more efficient. Indeed, the NC not only reduced the oxidative stress markers better but also provided more prolonged protection, probably due to its increased bioavailability and controlled release. This agrees with the results of Kalisz et al. [56] and Arshneshin et al. [17], who have further reported that phenolic nanoparticles improve oxidative stress tolerance by elevating the stability and activity of antioxidant systems during abiotic stresses. The inductive role of CA in reducing oxidative stress was also noticed under salinity stress conditions by Mughal et al. [55] and Mehmood et al. [54]. The activities of CAT and APX showed an appreciable increase in the presence of CA and CA-NCs, with the latter having a higher stimulation. This is in agreement with [43], who recorded that nanostructured phenolic compounds enhance enzymatic defenses more than their conventional ones. Besides, protease activity, a vital marker of protein degradation under stress, was better moderated with CA-NCs, thus meaning less protein damage compared to salt-stressed ones. The PPO activities, indicative of enhanced phenolic metabolism, were also significantly higher in both CA and CA-NCs treatments. Shahmarbiglou and Razavi [52] found similar observations and underlined the ability of nanocarriers to augment secondary metabolism pathways in stressed plants. Indeed, such nanocarriers, like CA-NCs, enhance the stability of polyphenolic compounds and increase their metabolic pathways, thereby leading to higher PPO and protease activities with improved tolerance to stress.

Applying CA and its NC form significantly enhanced the level of secondary metabolites, including flavonoids, phenolic compounds, tannins, and anthocyanins, under salinity stress. The enhanced production of flavonoids, anthocyanins, and tannins by CA-NCs under salinity stress (Fig. 6C-F) is likely mediated by the jasmonate signaling pathway. Jasmonic acid activates transcription factors such as MYB and bHLH, which regulate genes like PAL and CHS involved in phenolic and flavonoid biosynthesis [57]. The sustained release of CA from NCs may amplify these responses, boosting metabolite accumulation. Salinity alone increased these compounds, with the highest levels observed at 150 mM NaCl. However, such an enhancement was more pronounced with CA or CA-NCs, with the latter representing the most noticeable effect. Such synergistic effects of CA-NCs and salt stress are corroborated by recent findings from Shiri et al. [58] and Nanehkaran et al. [25], proposing that nanomaterials improve the bioavailability and activity of phenolic compounds and, subsequently, enhance plants’ tolerance to the applied stress more effectively than their conventional forms do. Enhanced flavonoids, phenols, tannins, and anthocyanins have been shown to contribute to better antioxidant capacity, thus preventing the plant from oxidative damage. The findings of Arshneshin et al. [17] have proven that nanocarriers enhance the accumulation of secondary metabolites, which may substantially improve plant resistance against various stresses. Interestingly, the highest increase of flavonoids, phenols, and anthocyanins, caused by CA-NCs at 150 mM NaCl, highlights their better performance at enhancing plant tolerance to salinity compared to previously related studies using other plants subjected to salinity stress [53]. This gives an idea of the immense potential for CA-NCs as an effective means of improving plant adaptability to abiotic stresses through antioxidant defense induction. CA-NCs’ superior performance, likely due to their 99.8 nm size, enhancing cellular uptake and bioavailability, aligns with nanoparticle properties described by Kumar et al. [59].

Conclusion

This study elucidates the superior mechanisms of CA-NCs over free CA in mitigating salinity stress in M. officinalis. The primary advantage lies in the nano-scale size (~ 99.8 nm) of CA-NCs, which enables more efficient penetration and interaction with plant tissues, leading to enhanced bioavailability. The key protective mechanisms involve a multi-faceted approach. Firstly, CA-NCs effectively reverse salinity-induced damage to the photosynthetic apparatus by restoring chlorophyll content and improving stomatal conductance, thus enhancing photosynthetic efficiency. Secondly, they function as potent antioxidants, directly scavenging reactive oxygen species. This is evidenced by a significant reduction in oxidative stress markers like H₂O₂ and MDA and a concurrent boost in the activity of native antioxidant enzymes. Furthermore, CA-NCs play a crucial role in osmotic adjustment. They better modulate stress markers by improving RWC and promoting the accumulation of osmolytes like proline, which helps maintain cellular turgor and integrity under stress. Finally, CA-NCs uniquely enhance the plant’s innate defense system by upregulating the synthesis of protective secondary metabolites, such as flavonoids and anthocyanins. This combination of efficient delivery, robust antioxidant action, osmotic protection, and defense activation makes CA-NCs highly effective in enhancing salinity stress tolerance. Their improved stability, bioavailability, and efficient delivery make them promising candidates for agricultural applications, particularly in mitigating the effects of abiotic stress.

Authors’ contributions

**L. Ranjbar** : Writing – original draft, Software, Visualization, Data curation, Methodology, Investigation, Formal analysis, Conceptualization. **S.M. Razavi** : Visualization, Software, Methodology, Writing – review & editing, Validation, Supervision, Data Curation. **A. Khalofah** : Software, Conceptualization. **A. Ghorbani** : Validation, Data Curation, Writing – review & editing, Conceptualization.

Funding

Not applicable.

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.