Silymarin orchestrates reactive oxygen species metabolism, ion homeostasis and morpho-physiological traits to confer salt tolerance in Brassica napus

Abstract

Considering the devastating effect of salt stress on crops the present study investigates the regulatory role of exogenous silymarin in enhancing antioxidant defense system and morpho-physiology of Brassica napus under salt stress. Twenty-three-day-old rapeseed (Brassica napus cv. BARI Sarisha-18) plants were supplemented with a foliar spray of 250 ppm silymarin followed by two doses of NaCl, viz. 75 and 150 mM. This growing condition was maintained for the following 30 days. Salinity resulted in reduced biomass production, growth attributes, and relative water content of rapeseed plants with increased levels of Na⁺ ions, hydrogen peroxide (H₂O₂), lipid peroxidation, electrolyte leakage (EL), and proline (Pro) content. This led to oxidative damage by suppressing the activities of antioxidant enzymes. Silymarin reduced lipid peroxidation, H₂O₂, EL, and Pro content by 23, 15, 9, and 17%, respectively in 150 mM NaCl-stressed plants compared to their corresponding controls. The activities of glyoxalase, as well as antioxidant enzymes such as ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, catalase, glutathione peroxidase, glutathione S-transferase, peroxidase, lipoxygenase, and superoxide dismutase were also upregulated due to silymarin application. Findings from the current study suggest that salt-induced rapeseed plants exhibited reduced growth attributes alongside elevated oxidative stress markers, including both enzymatic and non-enzymatic antioxidants. Furthermore, it highlights the potential of silymarin as a potential growth regulator and antioxidant that can enhance salt tolerance in rapeseed plants by reducing the harmful effects of reactive oxygen species.

Introduction

Salinity is a major stressor that reduces agricultural output in most parts of the globe. According to the Food and Agriculture Organization (FAO) of the United Nations, over 424 million hectares of topsoil and 833 million hectares of subsoil are salinized around the world¹. Even though, natural processes such as climate, pedology and lithology contribute to the salinization of the soil, human actions especially irrigation have significantly increased the process. Additional factors are over fertilization and pollution of the environment with chemical substances². Currently, salinity affects approximately 6% of the total land of the world and over 20% of irrigated land, reducing crop yields by up to 70% in certain instances^3,4. It is one of the significant challenges that limit crop productivity and promotes the buildup of reactive oxygen species (ROS), which causes significant harm to the structure of enzymes, nucleic acids, and lipids⁵. In addition, sodium and chloride ions in high concentrations disrupt the sodium potassium ratio, which impedes membrane potential, turgor and osmotic pressure as well as phototropisms⁶. The increased uptake of Na⁺ and Cl⁻ reduces the osmotic potential between soil and root solutions reducing water uptake. Also, high ion levels interfere with nutrient absorption⁷. Shabala and Munns⁸ noted that salinity disrupts chlorophyll structure and its integrity by negatively impacting photosynthesis by inhibiting the electron transport chain and causing oxidative stress. Plants need to improve their physiochemical reactions to sustain ion homeostasis, control oxidative injury, and perform detoxification procedures in salinity^9,10.

Silymarin is the primary bioactive compound isolated in Silybus marianum (milk thistle) and it is currently being applied to aid plant growth under environmental stresses to maintain mitochondrial activity and cellular redox status by enhancing enzymatic and non-enzymatic antioxidant pathways¹¹. Milk thistle is more likely to store more silymarin and its derivatives under severe drought conditions. This was associated with the amplified antioxidant enzyme activity and the upregulation of chalcone synthase gene expression that led to the enhancement of drought tolerance¹². Also, foliar application of silymarin-enriched bee-honey solution (SBH) has presented promising outcomes in enhancing growth and productivity in plants. This treatment not only decrease ROS buildup but also enhances physiological and biochemical characteristics, nutrient absorption, and antioxidant immunity. The addition of silymarin to bee-honey greatly enhances its biostimulatory activity, which qualifies it as a useful option for enhancing the health and productivity of plants in harsh environments^13,14. Silymarin and clove fruit extract increased wheat plants tolerance to Cd stress by enhancing different metabolic and physiological processes¹⁵. Salman et al.¹⁶ noted that various concentration of silymarin (10, 25, 50, 125, 250, 500, 1000 and 2000 mg L⁻¹) was one of the factors that induced rice blast disease resistance by Magnaporthe oryzae. Their findings show that silymarin 500 mg L⁻¹ decreased the severity of the disease to 23.5% in the green house and in the field.

Rapeseed (Brassica napus L.) is an important oilseed crop, which is grown with the purpose of obtaining high-quality vegetable oils as well as used as leafy greens, spice, and animal feed due to the high protein level¹⁷. In 2020 and 2021, the global B. napus production was around 74 million tons, and this is projected to increase in the next few years¹⁸. Rapeseed oil has a great demand in terms of nutrition. It is marked by the low content of undesirable saturated fatty acids and the high content of monounsaturated fatty acids. Moreover, it is a good natural source of bioactive compounds, such as vitamin E, phenolic acids, and phytosterols¹⁹. Rapeseed is a also a flexible industrial crop. Its seeds are being utilized in the manufacture of biofuels, adhesives, surfactants, and cosmetics²⁰. Although rapeseed is assumed to be moderately salt-tolerant²¹, salinity tolerance has been found to vary widely across cultivars, namely, in the early seedling phase²². Salinity is an osmotic and ionic stressor, which negatively influences the growth, photosynthesis, and nutrient uptake of rapeseed particularly during the initial stages of development, leading to a decrease in crop yield^23,24. Salinity also leads to increased electrolyte leakage (EL) and depletion of biomass and relative water content (RWC) in Brassica species^25,26.

So far, there are hardly any study on the effects of exogenously applied silymarin as a foliar spray on the physiological and biochemical functioning, photosynthetic performance, antioxidant defense systems and enzymatic activities of rapeseed subjected to salt stresses. Based on the existing literature^13,27,28, we supposed that cellular antioxidant capacity of the plant would be improved by foliar application of silymarin, which would reduce the adverse effect of salinity. This study was thus aimed at determining the effect of silymarin leaf treatment, as an activator of plants, on the growth and yield parameters, physiological and biochemical characteristics, in salt-stressed rapeseed plant.

Materials and methods

Plant materials and experimental design

To conduct this experiment rapeseed seeds (Brassica napus cv. BARI sarisha-18) were sourced from Bangladesh Agricultural Research Institute (BARI) in Joydebpur, Bangladesh. After sun drying and removing unwanted materials the seeds were sown in 14L pots containing soil prepared with vermicompost (1:1) and other fertilizers. At 23 days after sowing (DAS) foliar application of silymarin extract was applied, where tween-20 was added with the solution as a surfactant. We obtained this silymarin extract (80%, supplier: Healthy hey nutrition, India) from our collaborators and applied it directly in the pot experiment. Silymarin was applied four times at an interval of 7 days. For each application, 250 ppm silymarin solution was sprayed uniformly onto the plants until the foliage was fully drenched. The NaCl (75 mM and 150 mM) was also applied in four equal splits at 7-day intervals (23, 30, 37, and 44 DAS). In every time, the application volumes of NaCl were consistent. The NaCl was applied as a soil drench, and each pot contained about 12 kg soil. At 53 DAS a detailed assessment of both biochemical and morphological data was performed to evaluate plant responses. This experiment was conducted following a completely randomized design (CRD) with three replications.

Determination of plant shoot length, root length, SPAD value and biomass accumulation

The plant shoot length of randomly selected plants was measured by taking the measurement from the base of the plant to the apex of the leaf with a measuring scale. After the final measurement, the values were averaged. The root length was measured by pulling out the plants from the soil, these were cleaned off and measured with the measuring scale.

To find out the chlorophyll content (Chl) of leaf, fully expanded leaf samples were used. A soil and plant analysis development (SPAD) meter (FT GREEN LLC, Wilmington, DE, USA) was utilized over randomly selected samples from each set of treatment.

The stem diameter of plants was taken cautiously by a digital slide caliper, averaged and expressed in millimeters (mm).

After pulling out the whole plant sample carefully and removing excess dust the shoots and roots were separated and shoot fresh weight (FW) and root FW was taken. The samples were then kept in room temperature for reducing the initial moisture for some periods, then were transferred to the oven for removing the extra moisture at 80 ℃ of temperature for 72 h. The dry weight (DW) of the shoot and root were then measured.

Estimation of relative water content

The protocol by Barrs and Weatherley²⁹ was followed to quantify the RWC of leaves where the samples were cut from each set of treatment. These leaf cuttings were then weighed cautiously for the FW. The samples were then submerged in distilled water (dH₂O) overnight in a dark place at room temperature. After the time period, the samples were then taken out from the submerged condition. The extra moisture was drained off by using tissue paper and the turgid weight (TW) of leaves were taken and recorded. After that the samples were oven dried, later the DW was monitored. The final calculation was done performing the subsequent equation:

Estimation of chlorophyll fluorescence

The chlorophyll fluorescence of leaves was taken carefully by using Chlorophyll fluorescence meter (Pocket PEA, Hansatech Instruments Ltd., Norfolk, UK). Clips were attached to plant leaves to create a dark phase. After some time the fluorescence meter was used to record the data.

Determination of oxidative stress indicators

A standard protocol stated by Heath and Packer’s³⁰ was followed to assess the level of stress indicator malondialdehyde (MDA) content. The leaf samples were weighed about 0.5 g from each treatment and then homogenized appropriately with mortar and pestle maintaining chilled condition with 5% of 3 ml trichloroacetic acid (TCA) and then centrifuged at 11,500 × g. A clear supernatant was then obtained after centrifugation and then the supernatant was consolidated with 4 mL of thiobarbiturioc acid (TBA) reagent containing TBA (0.5%) and TCA (20%). These solutions were incubated for 30 min at a temperature of about 95 °C in a water bath. Later, samples were cooled at room temperature and spectrophotometric readings were observed at 532 nm and wavelength of 600 nm. The deduction of non-specific values was done properly and then the final computation was done by using an extinction coefficient (155 mM⁻¹ cm⁻¹) and expressed as nmol g⁻¹ FW.

Yu et al.³¹ method was used to quantify the level of hydrogen peroxide (H₂O₂) content. 0.5 g leaf samples were collected and ground in ice cooled mortar pestle with 3 mL of 5% TCA to acquire the supernatant. Then 1 mL solution was taken and consolidated with 1 mL buffer (pH 7.0) potassium-phosphate (K-P) and 1 mL potassium iodide solution and kept in room temperature in the dark. The absorbance was then read at 390 nm by a spectrophotometer. The H₂O₂ content was recorded in nmol g⁻¹ FW.

Estimation of electrolyte leakage

Fresh leaf samples (0.5 g) were taken for the estimation of EL, following the protocol of Dionisio-Sese and Tobita³². The samples were first washed for removing the dust particles and then chopped into small pieces. The pieces were then taken into falcon tubes with distilled water (dH₂O). After boiling the samples at 40 ℃ just for 1 h the initial electrical conductivity (EC₁) was recorded by an Electrical Conductivity (EC) meter. After taking the EC₁, the samples were autoclaved at a temperature of 121 ℃ and the final EC (EC₂) was taken. Following the equation given below the electrolyte leakage was computed:

Estimation of the proline content

Bates et al.³³ highlighted a standard method which was used to determine the proline (Pro) content. The freshly harvested leaf samples (0.5 g) were collected and macerated with 5 mL 3% aqueous sulfosalicylic acid in a chilled mortar and pestle after that the macerated solution was centrifuged at 11,500 × g just for 15 min to obtain a clear supernatant. After finishing the centrifugation, the obtained supernatant was dissolved with a series of chemical solutions including glacial acetic acid and acid ninhydrin which was then dissolved in 1 mL 6 M of phosphoric acid solution and incubated at a temperature of about 100 ℃ for 1h in a water bath. Then, the solution was cooled off in room temperature and toluene was added to that solution in adequate amount. The solution was then vortexed properly and the colored chromophore was monitored at 520 nm. Lastly the final calculation of Pro content was made using a standard curve. To prepare the standard curve for proline estimation, a stock solution of 1 mg ml⁻¹ is prepared by dissolving 10 mg of proline in 10 ml of water, which is then diluted to concentrations ranging from 10 to 80 µg ml⁻¹. For a 2 ml final volume, specific aliquots of the stock (40–320 µl) are mixed with distilled water (1960–1680 µl) to achieve the target gradients. These standards are reacted with acid-ninhydrin and glacial acetic acid in a boiling water bath, followed by extraction of the resulting chromophore into a toluene layer. Finally, the absorbance is measured at 520 nm using a spectrophotometer to plot a linear regression line, allowing for the quantification of proline in unknown samples.

Measurement of ion content

A compact Na⁺, K⁺ ion meter device (Laquatwin-K-11, HORIBA Advanced Techno Co., Romeoville, IL, USA) was used to find out the Na⁺, K⁺ ion contents of the leaves. The freshly harvested leaf samples were collected, with the help of a cell sap extractor. The cell sap was transferred to the sensor head of the device, which was previously calibrated. When the sensor displayed the values after 2–3 s, the Na⁺ and K⁺ ion contents were assured.

Quantification of ascorbate and glutathione pool

The quantification of non-enzymatic antioxidants like ascorbate (AsA) and glutathione (GSH) was assessed by following the standard method Kampfenkel et al.³⁴. According to his technique the leaf sample were freshly harvested and homogenized in chilled mortar and pestle through which about 3 mL of 1 mM ethylenediaminetetraacetic acid (EDTA) in 5% meta-phosphoric acid was used and sent it for centrifugation at 11,500 × g. After centrifugation a clear aliquot was obtained and it was then neutralized with 0.5 M K-P (potassium-phosphate) buffer pH 7.0, 0.1 M dithiothreitol (DTT). Then dH₂O was added to the aliquot and the neutralized aliquot was consolidated with several chemicals containing 100 mM of K-P buffer pH 6.5 and 0.5 units of ascorbate oxidase (AO), then spectrophotometric readings were monitored at 265 nm by a spectrophotometer. Total and reduced AsA were quantified using a standard curve, and dehydroascorbate (DHA) was calculated by subtracting reduced AsA from total AsA³⁵. To prepare the standard curve for ascorbic acid (AsA) estimation, a 1 mM stock solution was first created by dissolving 1.76 mg of ascorbic acid in 10 mL of 5% TCA. From this stock, a series of working standards with concentrations ranging from 80 to 400 µM are prepared by mixing specific volumes of the stock solution with 5% TCA. Each mixture is then supplemented with 300 µL of 0.5 M potassium-phosphate (K-P) buffer (pH 7.0) and thoroughly vortexed to ensure homogeneity. The resulting preparations are kept for one hour at room temperature to allow for proper stabilization before the assay is performed. Following incubation, the absorbance is measured to establish a linear relationship between concentration and optical density, facilitating the quantification of AsA in plant samples.

The GSH content was also determined after neutralization of the acquired supernatant consolidated with (0.5 M) K-P (potassium-phosphate) buffer (pH 7.0) where oxidized by 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) in the presence of glutathione reductase reduced with nicotinamide adenine dinucleotide phosphate (NADPH). At 412 nm the electrophoretic absorbance readings were measured. The neutralized extract was utilized with vinylpyridiene beside (K-P) buffer for oxidized glutathione (GSSG). A standard curve mentioned earlier was utilized to assess the total GSH and GSSG content through an acquainted concentration of GSH. This standard curve was set with 12, 16, 20, and 24 µg mL⁻¹ GSH with 5% of meta phosphoric acid, then after the values of GSSG were subtracted from total GSH to enumerate the GSH³⁶.

Enzyme extraction, determination of soluble protein and enzyme

About 0.25 g leaf samples were collected and homogenized with a chemical series containing an extraction buffer 1 mL 50 mM K-P buffer (pH 7.0), 100 mM potassium chloride (KCl), 5 mM β-mercaptoethanol, 1 mM L-ascorbic acid (Asc) and at the end 10% (w/v) glycerol. The maceration was done by using the chilled mortar and pestle. After homogenization the homogenized solution was then dispatched for centrifugation at 11,500 × g for 15 min the acquired supernatant was then preserved in chilled condition for determining the protein and antioxidant enzyme activity³⁶.

Following the protocol denoted by Bradford³⁷ protein concentration was determined. The Bradford reagent was prepared by an adequate amount of Coomassie brilliant blue (CBB G-250), 100 ethanol, and 85 ortho-phosphoric acid and adding sufficient amount of dH₂O. After that 5 μL supernatant and 5 mL Bradford reagent was incorporated and at 595 nm spectrophotometric absorbance were monitored. Following a standard curve of 5, 10, 15, 20 and 25 μL bovine serum albumin with 5mL Bradford reagent the protein was determined.

Ascorbate peroxidase (APX, EC, 1.11.1.11) activity was assessed by using the 15 mM K-P buffer (pH 7.0), 0.1 mM H₂O₂, 0.5 mM Asc and 0.1 mM EDTA. At 290 nm the spectrophotometric readings were monitored and the extinction coefficient value at (2.8 mM⁻¹ cm⁻¹) were applied³⁸.

Determination of dehydroascorbate reductase (DHAR, EC:1.8.5.1) activity was determined by the protocol of Nakano and Asada³⁸. The enzyme solution was consolidated with 2.5 mM reduced GSH, 0.1 mM EDTA solution, 50 mM of K-P buffer (pH 7.0), and 0.1 mM DHA. The spectrophotometric readings were measured at 265 nm and an extinction coefficient value of 14 mM^–1 cm^–1 was used.

The protocol by Hossain et al.³⁹ was used for determining monodehydroascorbate reductase (MDHAR, EC: 1.6.5.4) activity, by utilizing enzyme solution with 50 mM Tris–HCl used as a buffer (pH 7.5) solution, 1 unit of AO, 0.2 mM NADPH and 2.5 mM Asc. The spectrophotometer wavelengths were recorded at 340 nm by using an extinction coefficient value.

The glutathione reductase (GR, EC:1.6.4.2) activity was determined by dissolving the enzyme solution with 0.1 M K-P (pH 7.8) buffer solution, 1 mM oxidized (GSSG), 0.2 mM (NADPH) and 1 mM EDTA. The absorbance was monitored at 340 nm and the activity was calculated applying an extinction coefficient of about (6.2 mM⁻¹ cm⁻¹)³⁶.

The Catalase (CAT, EC: 1.11.1.6) activity was assessed by obtaining the enzyme solution consolidated with the chemicals 50 mM K-P buffer (pH 7.0) and 15 mM H₂O₂ and then the decreased absorbance was monitored at 240 nm by using the extinction coefficient of about 39.4 M⁻¹ cm⁻¹³⁶.

Protocol developed by Nahar et al.⁴⁰ was followed for the estimation of glutathione peroxide (GPX, EC:1.11.1.9) activity, utilizing K-P as buffer solution (pH 7.0), 1 mM EDTA, 1 mM sodium azide, 0.12 mM NADPH, 2 mM reduced glutathione (GSH), GR, H₂O₂ chemicals and enzyme solution. At 340 nm the optical absorbance was observed by applying the extinction coefficient at 6.62 mM⁻¹ cm⁻¹.

The glutathione S-transferase (GST, EC: 2.5.1.18) activity was estimated by using a chemical series consisting of 0.25 M K-P buffer (pH 6.5), 1.5 mM GSH, and 1 mM 1-Chloro-2,4-dinitrobenzene (CDNB). To measure this activity, the spectrophotometer was set on 340 nm and later the readings were observed. After that the value were utilized and enumerated with the help of extinction co-efficient (9.6 mM⁻¹ cm⁻¹)³⁶.

A method stated by Doderer et al.⁴¹ was used to determine the lipoxygenase (LOX, EC: 1.13.11.12) activity. The buffer solution K-P (pH 6.5) and linolenic acid substrate along with Tween-20 was utilized for this estimation. The spectrophotometer readings were monitored at 234 nm, later the computation was done with the extinction co-efficient (25 mM⁻¹ cm⁻¹).

Hemeda et al.⁴² proposed a standard protocol to find out the peroxidase (POD, EC: 1.11.1.7) activity. The determination was done by utilizing a chemical series like guaiacol, subjected to oxidation, consolidated with K-P as a buffer (pH 7.0) solution and H₂O₂. The spectrophotometric readings were then monitored at 470 nm where the extinction coefficient of 26.6 mM⁻¹ cm⁻¹ was employed.

Determination of superoxide dismutase (SOD, EC 1.15.1.1) activity was found out using the method stated by El-Shabrawi et al.⁴³. The chemical series containing of xanthine, xanthine oxidase, nitro blue tetrazolium chloride (NBT) along with potassium phosphate buffer (pH 7.0) was utilized. At 560 nm the spectrophotometric readings were observed and the obtained values were subsequently analyzed.

Determination of methylglyoxal, glyoxalase I and II

Estimation of methylglyoxal content (MG) was determined using the technique of Wild et al.⁴⁴. About 250 mg of leaf samples were freshly harvested and then macerated with 5% perchloric acid. Later centrifugation was done at 11,500 × g just for 12 min, the centrifugation procedure was repeated for the extracted supernatant with charcoal materials. The saturated sodium carbonate solution was then used for neutralization of the acquired supernatant. The neutralize solution was then mixed with sodium-phosphate (Na-P) buffer solution along with N-acetyl-L-cysteine and kept for 15 min to form N-α-acetyl-S-(1-hydroxy-2-oxoprop-1-yl). At 288 nm the spectrophotometric readings were monitored and later the MG content was enumerated by using a standard curve.

Quantification of glyoxalase I (Gly I, EC: 4.4.1.5) activity was performed following the standard method stated by Hasanuzzaman et al.³⁶ with 100 mM K-P as buffer solution (pH 7.0) mixed with 16 mM magnesium sulfate, 35 mM methylglyoxal and 100 mM GSH along with enzyme supernatant. At 240 nm wavelength spectrophotometric readings were taken and then Gly I was enumerated.

Determination of glyoxalase II (Gly II, EC: 3.1.2.6) activity was done by conforming the technique previously stated by Hasanuzzaman et al.³⁶. The enzyme samples were consolidated with 100 mM Tris–HCl buffer pH 7.2, 0.2 mM DTNB, 1 mM S-ᴅ-lactoylglutathione (SLG). At 412 nm of spectrophotometric wavelength the Gly II activity was calculated where an extinction coefficient (13.6 mM⁻¹ cm⁻¹) was utilized.

Statistical analysis

The CoStat v.6.400 (2008) software was used for the analysis of variance (ANOVA) of several parameters. After that the means were compared by the Tukey honest significant difference (HSD) test at 5% probability. Correlation metrix was generated by Origin Pro 2022 (OriginLab, Northampton, MA, USA).

Results

Silymarin reduces salt induced growth inhibition in rapeseed

The shoot length of plants decreased by 11 and 29% respectively under NaCl stress at 75 and 150 mM, compared to the unstressed plant. Silymarin increased the shoot length by 11% under 150 mM salt stress compared to the stressed plant that were not treated with silymarin (Fig. 1A). Similarly, the root length also declined by 11 and 23% under both doses of salt stress while the values increased by 17 and 18% respectively by the supplementation of silymarin in comparison with the stressed plants (Fig. 1B). Additionally, the stem diameter of plants was also reduced by 15 and 30% under different doses of NaCl but silymarin application slightly improved the value (Fig. 1C). Figure. The SPAD value remained statistically similar across all treatments (Fig. 1D).

Fig. 1.

Changes in (A) shoot length, (B) root length, (C) stem diameter, and (D) SPAD value of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

The shoot FW declined by 31 and 59% respectively under both doses of salt stress in comparison with the control plants. On the other hand, the supplementation of silymarin increased the percentage by 17 and 15% compared to the stressed plant (Fig. 2A). Similarly, the shoot DW was also reduced considerably by 31 and 57% under both salt stress while silymarin supplementation increased the value by 24 and 20% respectively (Fig. 2B).

Fig. 2.

Changes in (A) shoot fresh weight, (B) shoot dry weight, (C) root fresh weight, and (D) root dry weight of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Improvement of relative water content and photosystem II efficiency by silymarin under salt stress

The RWC of leaf decreased by 19 and 24% under 75 and 150 mM NaCl stress in comparison with the control plants. The silymarin application slightly improved the relative water content compared to the unsupplemented salinity treated plant (Fig. 3A). Similarly, the chlorophyll fluorescence was reduced in stressed plants. The supplementation of silymarin slightly improve the percentage (Fig. 3B).

Fig. 3.

Changes in (A) relative water content, and (B) Chlorophyll fluorescence of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Reduction of oxidative stress markers, electrolyte leakage, and proline content by silymarin under salinity

The MDA content increased by 161 and 216% under 75 and 150 mM salt stressed plant in comparison with the control plant, while the supplementation of silymarin lowered the level by 25 and 23% respectively compared to the salt stress (Fig. 4A). Similarly, the H₂O₂ content escalated in salt stressed plant by 105 and 163% under different doses of salt stress in comparison with the control plant, whereas a reduction was noticed with the application of silymarin by 28 and 15% respectively compared to the stressed plant (Fig. 4B). The EL percentage also seemed higher in stressed plant by 14 and 27% compared to the control plant but in silymarin supplemented plant the percentage was lowered by 13 and 9% respectively in comparison with the stressed plant (Fig. 4C). Moreover, an increase was noticed for proline content in plant having 75 and 150 mM salt stress by 86 and 107% respectively in comparison with the unstressed plant, while the level was diminished by 16 and 17% with the application of silymarin compared to the stressed plant (Fig. 4D).

Fig. 4.

Changes in (A) MDA content, (B) H₂O₂ content, (C) electrolyte leakage, and (D) proline content of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Effect of silymarin on Na⁺ and K⁺ homeostasis under salinity

A disruption was noticed in ion homeostasis where the Na⁺ ion accumulation was higher by 145 and 250% respectively under 75 and 150 mM NaCl stress, compared to the control. After silymarin application the accumulation rate was lowered by 26 and 15%, respectively in comparison with the control plant (Fig. 5A). Similarly, K⁺ ion accumulation decreased in stressed plants by 19 and 32% under both salt stress but silymarin application increased the accumulation rate at 75 and 150 mM NaCl stress (Fig. 5B). Further looking at the Na⁺/K⁺ ratio, it escalated by twofold and fourfold under 75 and 150 mM salt stress, compared to the control plant while silymarin supplementation reduced the value by 37 and 26% respectively in comparison with the stressed plant (Fig. 5C).

Fig. 5.

Changes in (A) Na ⁺ content, (B) K⁺ content, and (C) Na⁺ /K ⁺ ratio of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Effect of silymarin on ascorbate glutathione pool under salinity

A significant decline in AsA content was observed under both levels of salt stress, decreasing by 47 and 52%, respectively, compared to the unstressed plants (Fig. 6A). On the other hand, the DHA content increased significantly under salinity, escalating by 148 and 187% relative to the control plants (Fig. 6C). However, the application of silymarin boosted AsA content by 83 and 64% while declining DHA content by 47 and 20% under 75 mM and 150 mM salt stress, respectively, compared to the stressed plants alone. Finally, the AsA/DHA ratio decreased by 79 and 82% under 75 mM and 150 mM NaCl stress, respectively, but silymarin treatment restored the ratio by sharply increasing it by 247 and 109% compared to the salt-stressed plants (Fig. 6E).

Fig. 6.

Changes in (A) AsA content, (B) GSH content, (C) DHA content (D) GSSG content (E) AsA/DHA ratio (F) GSH/GSSG ratio of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

The GSH level declined by 12% at 75 mM salt stress and by 41% at 150 mM NaCl stress compared to the control plant. However, the GSH level was escalated by the application of silymarin by 18 and 31% in comparison with the control plant (Fig. 6B). On the contrary the GSSG content were sharply higher by 74 and 164% respectively under both doses of salinity and the supplementation of silymarin sharply reduced the level by 39% at 75 mM salt dose and 16% at 150 mM salt compared to the stressed plant (Fig. 6D). The severity of salt stress considerably declined the GSH/GSSG ratio by 53 and 78% respectively than the control plant while it was restored by 124 and 46% by the application of silymarin in comparison with the stressed plant (Fig. 6F).

Silymarin regulates antioxidant and lipoxygenase activities in response to NaCl stress

The activities of APX, DHAR, MDHAR decreased by 57, 21 and 32% at 150 mM salt stress while the GR activity sharply escalated by 163 and 146% respectively under 75 and 150 mM salt stress compared to the unstressed plant. However, the APX activity was boosted by 84% after the application of silymarin under the 150 mM salt stress while silymarin application significantly increased the DHAR, MDHAR activity by 87 and 93%, 75 and 179% under 75 and 100 mM salt respectively compared to the stressed plant (Fig. 7A, B, C). On the other hand the GR activity was reduced by 34 and 35% respectively in comparison with the stressed plant by the application of silymarin (Fig. 7D).

Fig. 7.

Changes in the activities of (A) APX, (B) DHAR, (C) MDHAR and (D) GR of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

The CAT activity declined by 61 and 62% respectively at 75 and 100 mM salt stress compared to the control plant (Fig. 8A). The silymarin application improved its value by 233% at 75 mM and 147% at 150 mM salt stress. The GPX activity slightly increased by 35 and 19% respectively, compared to the control plant but supplementation of silymarin increased the level by 158 and 75% in comparison with the stressed plant (Fig. 8B). Similarly, the GST and POD level were also boosted by the application of silymarin by 124 and 116%, and 119 and 27% respectively, compared with the unsupplemented stressed plant (Fig. 8C, E). The SOD activity was lowered by 14% under 75 mM salt stress but its level was higher in 150 mM salt stress plant by 42% respectively compared to the control plant. After silymarin application the SOD level was escalated by 49% in 75 mM salt treated plant and 273% in 150 mM salt-imposed plant compared to only salt-imposed plant (Fig. 8F). Additionally,the LOX activity also increased by 50 and 124% respectively in comparison with the control plant. While a de-escalation was noticed under supplementation of silymarin by 23 and 18% respectively in comparison with unsupplemented but stressed plant (Fig. 8D).

Fig. 8.

Changes in the activities of (A) CAT, (B) GPX, (C) GST, (D) LOX, (E) POD, and (F) SOD of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Effect of silymarin on the glyoxalase system under salinity

It was also evident that the MG content was increased significantly by 88% at 75 mM salt stress and 112% respectively under 150 mM of NaCl stress in comparison with the control plant (Fig. 9A). However, the silymarin application significantly lowered the MG content by 27 and 23% respectively at 75 and 150 mM salt stressed plant. On the other hand, the Gly I and Gly II level were declined sharply by 26 and 30%, and 38 and 54% due to 75 and 150 mM NaCl respectively, in comparison with the unstressed plant but application of silymarin altered the level, by increasing it by 49 and 73%, and 33 and 45% respectively, compared to the salt treated plants (Fig. 9 B, C).

Fig. 9.

Changes in the activities of (A) MG content, (B) Gly I, and (C) Gly II of rapeseed under 75 and 150 mM salt stress and with the presence and absence of silymarin spray. The data are expressed as mean ± standard deviation from three replications (n = 3). Different letters on the bars indicate significant differences between treatments at p ≤ 0.05 based on Tukey’s HSD test.

Correlation matrix highlights relationships among stress markers, antioxidants, and growth traits

The correlation matrix was studied to see the various parameters that justify the responses of rapeseed, under the influence of salinity, with or without interaction of silymarin. This correlation clearly illustrates a strong relation among growth, physiological, oxidative stress and antioxidant parameters. Here, growth parameters including shoot length, root length, stem diameter, SPAD, fresh weight, dry weight, and relative water content demonstrated a strong positive correlation with each other. On the other hand, MDA, H₂O₂, electrolyte leakage, sodium ions, Na⁺/K⁺ ratio, along with MG were negatively correlated with the growth parameters, although they were positively correlated among themselves, which indicates the stress impedes plant development. (Fig. 10). Among the antioxidant enzymes only GR showed a positive correlation with the oxidative stress markers. Upon considering the non-enzymes, AsA and GSH were negatively interlinked while, DHA and GSSG exhibited a positive correlation. Additionally, plant growth attributes, RWC and Gly II was negatively affected by the salinity induced oxidative stress indicators.

Fig. 10.

Correlation matrix among the different parameters of rapeseed as affected by salinity. Here, SPAD—soil and plant analysis development value; FW—fresh weight; DW—dry weight; RWC—relative water content; Fv/Fm—chlorophyll fluorescence; MDA—malondialdehyde; H₂O₂—hydrogen peroxide; EL—electrolyte leakage; Pro—proline; Na⁺—Sodium ion; K⁺—potassium ion; AsA— ascorbate; GSH— glutathione; DHA—dehydroascorbate; GSSG— glutathione disulfide; APX—ascorbate peroxidase; DHAR—dehydroascorbate reductase; MDHAR—monodehydroascorbate reductase; GR—glutathione reductase; CAT—catalase; GPX—glutathione peroxidase; GST—glutathione S-transferase; LOX—lipoxygenase; POD—peroxidase; SOD—superoxide dismutase; MG—methylglyoxal; Gly I—glyoxalase I; Gly II—glyoxalase II.

Discussion

Soil salinization is becoming a major worldwide problem for sustainable agriculture, and it notably affects cellular and physiological disorders at all stages of plant development, thus reducing crop productivity⁴⁵. Excessive salinity affects the growth of plants, reducing uptake of essential mineral nutrients like K⁺ ion due to high accumulation of Na⁺ ion content of plant tissue⁴⁶. In this experiment, higher salt stress exerted a detrimental effect on the various growth attributes of the rapeseed plants including root and shoot lengths, stem diameter and fresh and dry weights of the root and shoot. Additionally, photosynthesis related parameters (SPAD value and Fv/Fm) decreased, which also indicates that salinity inhibited cell division and cell expansion. The most noticeable correlation was observed between growth and biomass reduction with the increased levels of Na⁺ ion and excessive H₂O₂ production. Increased H₂O₂ levels disturbs the integrity of the plasma membrane, causing ionic disequilibrium and suppression of vital metabolic reactions, which ultimately resulted in deteriorated plant growth⁴⁷. Exogenous supplementation of silymarin reduced salinity’s deleterious effects on stressed plants’ growth parameters. The significance of silymarin is demonstrated by recent reports of beneficial changes in different plant development, biochemical characteristics, and the plant defense system such as nonenzymatic antioxidants and antioxidative enzymes, following treatment of plants with silymarin alone under different abiotic stressors^12,13,15. However, the current study suggests that silymarin functions as an active growth regulator, enhancing the salinity tolerance of plants.

Silymarin is extracted from all parts of Silybum marianum (milk thistle) which is a medicinal plant and its extract consists of six major flavonoids and flavonolignans: silydianins, silychristins, isosilychristins, silybinins or silibinins (A and B), isosilybins (A and B) and flavonoids like taxifolin^11,48. These ingredients of silymarin help to enhance root architecture and function, ultimately enhancing water and nutrient uptake, growth, and biomass accumulation under salt stress⁴⁹. The Chl fluorescence parameter Fv/Fm is a measure of the maximal quantum efficiency of PSII. In this experiment, Fv/Fm was reduced by salinity and this showed that PSII system was damaged. Nevertheless, the silymarin was significantly recorded a higher in the Fv/Fm and SPAD parameters, which indicate that it stimulates protection of the photosynthetic pigments and function in Chl fluorescence and thereby it encourages plant growth ability under stress same as reported by Alharby et al.¹³. Besides having the mitigating effect on salinity, the application of silymarin also changed various physiological and biochemical parameters under the control circumstances, which demonstrates its biostimulating property beyond protecting stress. Previous research has shown that silymarin is capable of improving the plant growth, photosynthetic and antioxidant potential even in non-stressed plants, although the modification is typically less significant as compared to when plants are stressed¹³. These reactions can be caused by enhanced metabolic activity and stabilization of photosynthetic pigments, as well as increase of enzymatic and non-enzymatic antioxidant systems¹⁵. Equivalent growth and physiological functioning enhancements have been indicated in chili pepper and Atriplex nummularia upon the use of silymarin under normal circumstances (Abou-Sreea et al. 2021; ^50,51).

Salinity disturbs ion homeostasis in plants, mainly leading to Na⁺ ion over accumulation in the tissues. The accumulation interferes with the absorption and transport of K⁺ ion and other necessary nutrients from the roots to the leaves, resulting in nutrient deficiency and physiological imbalance⁵². Elevated Na⁺ ion concentrations and low K⁺ ion levels in photosynthesizing leaves can lead to chlorophyll degeneration and decreased thylakoid integrity, which could negatively affect PSII efficiency⁵³. Thus, the sequestering Na⁺ in salt sensitive tissues, mainly in the shoots, is a key factor for salt tolerance in plants⁴⁷. In this study, application of higher NaCl concentrations led to a significant increase in Na⁺ and decrease in K⁺ contents in the leaves of rapeseed. Such alterations resulted in higher Na⁺ /K⁺ ion ratio which is a crucial matter for ionic imbalance index and stress severity. Similar result has also been described in other crops like Oryza sativa⁵⁴, Glycine max⁵⁵, Zea mays⁵⁶, as well as Barssica napus⁵⁷. High salinity may also raise the rhizosphere pH, leading to a decrease in the levels of available ions such as K⁺. Furthermore, K⁺ and Na⁺ share common binding sites involved in cellular activities, such as protein synthesis and enzyme activation, repression of K⁺ uptake induced its deficiency⁵⁸. However, in this study, by lowering the levels of Na⁺ in the stressed plants’ leaves, the external application of silymarin considerably mitigated these negative effects. In this research, lowering of Na⁺ as a result of silymarin foliar spray was accompanied by enhancements of K⁺ in leaves, the observed decrease in Na⁺/ K⁺. This indicates that silymarin contributes to the maintenance of ionic homeostasis, which is important for plants to adapt to salt stress.

Osmotic stress due to salinity strongly inhibits the water absorption of plants by lowering the water potential in the root zone. This situation is compounded by a decrease in stomatal opening and hence a restriction in the extent of transpiration driving water transport⁵⁹. Salinity imbalances ionic equilibrium and causes water deficiency in plant cells, leading to physiological changes such as decrease in turgor pressure, shrinkage of the plasma membrane, and changes in cell wall architecture⁶⁰. These changes are harmful to cell stability and function, which is more likely to suppress plant growth in saline environments. To combat with osmotic stress, plants active complex signalling pathways which moderate gene expression patterns, biosynthesis of osmolytes and water transport functions⁶¹. Osmolytes including Pro, polyols, and sugars contribute significantly to decrease osmotic potential in the cytoplasm to retain osmotic balance. These molecules are also signaling agents that regulate the concentration of abscisic acid (ABA), the expression of stress-related genes and cellular adaptation to saline stress⁶². The RWC significantly decreased, while Pro contents significantly increased due to the rising salinity levels in the present study, which aligns with previous reports of rapeseed^3,4,59,63,64. In this experiment, the exogenous application of silymarin also reduced the Pro accumulation and minimized the negative effects of osmotic stress, this could be attributed to the higher RWC under saline conditions. These findings are in consistent with Alharby et al.¹³, who found that silymarin application resulted in an enhanced RWC in Cd-stressed maize plants. Silymarin decreased 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity, which enhances RWC and helps to minimize osmotic damage in plants by maintaining cellular hydration and membrane integrity⁶⁵. These findings indicate that exogenous application of silymarin helps to mitigate osmotic stress under salt stress.

When ROS builds up, the redox balance is disrupted and plant cells suffer through oxidative stress. Salinity induces both osmotic and oxidative stress, which cause detrimental effects on growth of plants by increasing the accumulation of lipid peroxidation, H₂O₂ production, denaturation of proteins and amino acids as well as the oxidation of DNA and RNA due to insufficient antioxidant activity⁶⁶. When membrane-associated enzymes are involved and oxygen molecules are reduced, ROS accumulates excessively. Lipid peroxidation or elevated MDA level as a result of unsaturated fatty acid peroxidation within the membrane is caused by ROS. Ionic imbalance disrupts mitochondrial respiration which elevates ROS generation. Damage to the permeability of cell membranes causes EL from leaves⁶⁷. This study found that MDA, H₂O₂, EL, and LOX levels were enhanced under salt stress conditions, which is consistent with earlier findings for rice⁶⁸, pea⁶⁹, and maize⁷⁰. But, supplementation with silymarin alleviate the level of MDA, H₂O₂, EL, and LOX under different level of salt stress. Flavonolignans (like silibinin, isosilybins) from silymarin extract interact directly with lipid bilayers, scavenge lipid radicals, and terminate chain reactions of lipid peroxidation, they also stabilize membrane integrity, reduce EL caused by peroxidative damage⁷¹. Taxifolin and silychristin from silymarin act as direct scavengers of H₂O₂. Additionally, silibinin upregulates antioxidant enzymes like CAT and POD, which decompose H₂O₂ into water and oxygen⁷². Likewise, silibinin activates Nrf2 pathway which increases transcription of antioxidant gene such as CAT⁷³. In previous studies, according to El-Sappah et al.¹⁵, silymarin reduced oxidative stress of wheat under Cd stress, similar result also found in maize under similar stress¹³.

Upregulation of AsA-GSH pathway can scavenge the over accumulation ROS. In our present study, salinity downregulated the AsA content and upregulated the DHA content, which ultimately decreased the AsA/DHA ratio. Furthermore, downregulation of GSH and upregulation of GSSG content, resultant in decreased GSH/GSSG ratio. Similar findings were observed in salt exposed rapeseed plants^59,63. Salt stress alters the activity of the enzymes involved in the AsA-GSH pathway. In the Asada-Halliwell or AsA-GSH pathway, for detoxifying H₂O₂ into H₂O, APX receives an electron from AsA and gets oxidized to MDHA. Later, formation of AsA and DHA occurs due to the antioxidant activity of MDHAR by using NADPH as a reducing agent. Following that, the partially produced DHA converts to AsA with the aid of GSH. Afterwards, reductant NADPH donates an electron and with the assistance of GR, GSSG is converted to GSH⁷⁴. However, the application of silymarin increased the AsA/DHA and GSH/GSSG ratio which indicated better salt stress tolerance. The flavonolignans present in silymarin promotes the antioxidant activity by breaking the chain reactions of free radicles⁷⁵. According to previous literature, foliar application of silymarin enriched solution alleviated the salt induced stress in seedlings of Atriplex nummularia by enhancing the activity of AsA and GSH⁵¹. Similar findings also observed in wheat (Zea mays) when exposed to cadmium stress, as the active ingredients can chelate the heavy metal/metalloid toxicity by enhancing the antioxidant defense and productivity¹¹.

For balancing redox homeostasis and ROS detoxification both non-enzymatic and enzymatic antioxidant enzymes play a crucial role in the antioxidant defense system. Salinity exposed plants altered the enzymatic activity to maintain the redox balance. Antioxidant enzymes CAT, GPX, GST, LOX, POD, and SOD are also engaged in this mechanism similar to the APX, DHAR, MDHAR, and GR, which are engaged in the AsA-GSH pathway. In our recent study the activity of GPX, GST, LOX, and POD increased but the activity of CAT and SOD decreased under salt stress. Initially, SOD converts the toxic superoxide anion (O₂^•−) into H₂O₂, later in the cellular compartment formation of H₂O and O₂ occurs from H₂O₂ by the antioxidant activity of CAT. Glutathione as an electron donor facilitate the quenching of H₂O₂ and conjugation with xenobiotic and electrophilic compounds, where GSH aids as a substrate to elevate the activity of GPX and GST to ameliorate the ROS toxicity³⁶. Plants subjected to salt stress showed an increment of MDA content which indicates lipid peroxidation in the cellular components. Lipoxynase catalyzes the lipid peroxidation in a two-step process. In stressed plants the substrates of LOX, polyunsaturated fatty acids and polyunsaturated fatty acid hydroperoxide increased⁷⁶. Minimizing cellular damage by catalyzing H₂O₂ and other phenolic compounds is led by POD activity in the plants⁷⁷. Cell wall lignification and regulating the over accumulation of H₂O₂ in the subcellular compartments is a pivotal role played by POD. Safikhan et al.⁷⁸ reported that milk thistle itself may have some antioxidant activity which protects the plants from salinity induced stress by altering the intercellular concentration of the ions Supporting this assumption, drought stress induced the accumulation of bioactive compound silybin, which increased enzymatic activity in turn oxidative stress¹². In another study, foliar application of silymarin in wheat, elevated the Cd stress resistance by upregulating the CAT, POD, and SOD activity¹⁵. Maize plants subjected to Cd stress when stimulated with silymarin modulated the oxidative stress by enhancing the activity of APX, GR, CAT, and SOD¹³.

Cytotoxic compound MG is a byproduct from triose phosphate in the glycolysis pathway. Accumulation of MG also initiates due to the activity of MG synthase. Low concentration of MG in plant cells is a normal phenomenon but the over generation of MG inhibit cell proliferation, induce oxidative stress by promoting ROS accumulation. But plant has their own mechanism to scavenge the toxic effect of MG in the cellular level⁷⁹. Plants can prevent the toxicity by the glyoxalase system, where Gly I and Gly II utilize GSH as a cofactor and catalyzes MG to protect plant from the oxidative stress. Initially formation of SLG occurs by the catalyzation of Gly I which later Gly II catalyzes to D-lactic acid. In this study salt stress accelerates the formation of MG but supplementation of silymarin confers the salt stress tolerance by upregulating the Gly I and Gly II content⁸⁰. Flavonoids present in the extract of silymarin can scavenge free radicles as they have the antioxidant activity and protect the plants from oxidative stress¹². Antioxidant gene expression, transcription of related genes, enhancement of hormonal homeostasis improved when silymarin enriched biostimulants was applied. Glutathione present in silymarin extract modulated the phytohormones and eliminated the toxic free radicles¹³. Increment of the antioxidant defense system promots the detoxification of MG in our findings. All things considered, silymarin has the potential to boost growth, water usage efficiency, scavenge ROS, and activate antioxidative and photoprotective defense mechanisms (Fig. 11).

Fig. 11.

Schematic representation of the mechanism underlying salt stress tolerance in B. napus plants induced by the application of silymarin.

Despite such encouraging results, there are several limitations that should be acknowledged. The current study was primarily based on physiological and biochemical markers, and the subsequent molecular mechanisms were not confirmed by analyzing the expression of genes, protein activity, or omics. Consequently, the regulatory mechanisms that are related to salt tolerance upon silymarin are still partially appreciated. Moreover, the experiment was carried out in controlled pot condition, which does not necessarily represent the complicated interactions that take place in natural field conditions, such as heterogeneity of the soil, variability in climate, and chronic stress exposure. Moreover, the best dosage, application time and long-term performance of silymarin on crop production and soil–plant interactions need additional testing. This needs to be supplemented with transcriptomic, proteomic, and metabolomic studies and field-scale experiments that will prove the action of silymarin and determine the agronomic viability of silymarin as a sustainable biostimulant to enhance salinity tolerance in crops.

Conclusion

This study investigated the impact of saline soil on rapeseed growth, along with a range of physiological and biochemical indicators. Our results showed that salt stress reduced mustard growth related traits, whereas oxidative stress markers, both enzymatic and non-enzymatic antioxidants, and redox status were elevated compared to plants not exposed to stress. However, foliar application of silymarin proved to be an effective and practical approach for alleviating the harmful effects of salt stress. Silymarin-treated rapeseed plants demonstrated improved tolerance to the examined salt doses by reducing ROS accumulation through the upregulation of various antioxidants (AsA, APX, DHAR, MDHAR), along with lowered levels of pro, MDA, H₂O₂, EL, DHA, GSSG, and LOX activity. In conclusion, our findings indicate that foliar application of silymarin can be effectively utilized to boost salt stress tolerance and enhance the development of Brassica sp. under saline soil conditions. Further in-depth studies are needed to gain a clearer understanding of the specific defense mechanisms activated by Silymarin that contribute to enhanced stress tolerance in exposed plants.

Author contributions

Mirza Hasanuzzaman: conceptualization, Investigation, Formal analysis, Methodology, Supervision, Resources, Writing—review and editing. Samiul Alam: investigation, Visualization, Writing—original draft. Md. Sarwar Hosen: investigation, Data curation, Visualization, Writing—original draft. Samiha Rummana: formal analysis, Writing—original draft, Methodology. Md. Mahabub Alam: conceptualization, Methodology, data curation, Validation, Supervision.

Funding

This research did not receive any specific grant from funding agencies.

Data availability

All data generated or analyzed during this study are included in this article.

Declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ethics approval and consent to participate

No endangered or legally protected species were used. The plant material, Brassica napus cv. BARI Sarisha-18 is a popular variety widely cultivated in Bangladesh. Therefore, no permission is required. The seeds were collected from the Oilseed Research Center of the Bangladesh Agricultural Research Institute (BARI). The plant material used for this study was in compliance with local and national guidelines. This study needs no further affirmations.

Consent to publish

Not applicable.