1 Introduction

Increasing heavy metal (HM) levels created by human industrial activities have led to rising soil and water contamination (Singh et al. 2021). HMs that are not biodegradable and can stay in the environment for a long time include arsenic (As), cadmium, chromium, mercury, lead, and zinc, making soils unfit for cultivation (Shah and Daverey 2020). Among the numerous human-made activities that have led to environmental contamination include the usage of high-As agricultural paste, fertilizers, untreated wastes, fossil fuels, and geological processes (Alam et al. 2019; Siddiqui et al. 2020). Because As has mutagenic qualities and is a common metalloid pollutant, it impacts all types of life in all biomes (Zemanová et al. 2021).

Arsenic’s toxicity varies depending on its many forms, and arsenite (AsIII) and arsenate (AsV), in particular, are more toxic than organic forms (Bhat et al. 2021). Plants uptake AsIII through several nodule-26-like intrinsic proteins, whereas AsV enters the plant cell via various inorganic phosphate (iP) channels (Abbas et al. 2018). According to the authors, AsV shares several chemical properties with iP molecules, which can interfere with some P-dependent metabolic processes during adenosine triphosphate production. In contrast, AsIII interacts with sulfhydryl groups, blocking several cellular biochemical activities and enzymatic processes (Coelho et al. 2020). Plants are affected by As in both morphological and physiological ways. Effects of As toxicity on plant growth include leaf abscission, membrane damage, increased oxidative stress, impairment of protein biosynthesis systems, inhibition of root hair growth, and decreased chlorophyll and transpiration efficiency (Beniwal et al. 2023). Because of this, As-induced oxidative stress may be identified utilizing reactive oxygen species (ROS) accumulation measures and signs of the scavenging process, such as antioxidant systems.

Several methods have been applied for removing HM pollutants from the environment, but they have drawbacks such as logistical issues, long periods, high costs, and mechanical complexity (Sumiahadi and Acar 2018). Several plant extracts are applied as effective natural biostimulants to promote plant growth under diverse environmental stress. Biostimulants are physiologically active ingredients that can boost a plant’s tolerance to environmental stress by stimulating activities, including biomass production, photosynthesis, nutrient uptake, and flowering (Sharma et al. 2014). Nevertheless, according to the Package (2016), biostimulants can be broadly divided into two groups non-microbial and microbial. These two groups can then be further subdivided based on the nature of the biostimulants, formulation, mode of action, usage, kind of action, and beneficial effects (Rouphael and Colla 2018). Plant biostimulants are elements from nature that cause physiologic and molecular reactions that affect the quality and yield of crops, even though their main objective fails to provide nutrients or defend plants (Rouphael and Colla 2018).

A well-known biostimulant known as humic acid (HA) has functional groups with oxygen, including alcoholic, carboxyl, carbonyl, phenolic hydroxyl, and methoxyl groups (Cozzolino et al. 2016). HA is a significant organic component of soil and is produced when organic matter, such as dead animal and plant remains, decomposes (Ai et al. 2020). Furthermore, HA can accelerate plant growth by making cell membranes more permeable and facilitating the flow of nutrients (Canellas et al. 2015). HA has an impact on plant growth in both direct and indirect ways. Directly, it increases plant respiration, hormonal growth responses, chlorophyll content, the permeability of membranes, etc. Indirectly, it improves the biological, physical, and chemical soil conditions (Vanitha and Mohandass 2014). The oxygen-containing functional groups of HA can interact with HM ions like As in the environment to facilitate their chelation and migration by complexation and adsorption (Qiao et al. 2019).

Moringa oleifera is a member of the Moringaceae family and is the most well-known medicinal plant as various parts of its tree contain many antioxidants, including ascorbate, β-carotene, flavonoids, and phenolics (Dhakad et al. 2019). M. oleifera leaf extract (MLE) was described as a biostimulant that boosts the growth and yields of several plants (Maishanu et al. 2017). MLE has a strong prospect for reducing metal toxicity because it is abundant in antioxidants (Kerdsomboon et al. 2021). According to studies by Amorim et al. (2023), M. oleifera seeds and leaf tissue could be applied as a biological absorbent to remove several HMs and metalloids from contaminated water.

HM environmental pollution is one of the main issues in Egypt (Al Naggar et al. 2018). Unfortunately, most Egyptian coastal zones along the Mediterranean Sea are disposed to heavy discharges of pollutants from countless anthropogenic activities (Dowidar 1988). According to Salman et al. (2019), As levels in soil samples from the Orabi area, which represents the eastern part of El Obour City, were extremely high. Additionally, during the movement and development of soil, As may have come from rocks in the Eastern Desert, where some serpentine rocks have As concentrations of up to 75 mg kg−1 (Sadek et al. 2015). In Assiut, the northern part of the governorate was found to be highly contaminated with HMs from human activities, including As (15.6 µg g−1), Cr (121.7 µg g−1), Co (37.7 µg g−1), Cu (33.9 µg g−1), Pb (39.2 µg g−1), and Ni (97.7 µg g−1) (Salman et al. 2021). The authors also noted that these figures were 1.5–5.0 times higher than the background levels observed globally in sediments and agricultural soil from Egypt.

One of the vegetable species that are most commonly grown and consumed worldwide is broccoli (Brassica oleracea) (Pizetta et al. 2005). Broccoli contains significant amounts of organosulfur compounds that encourage the activation of enzymes needed for carcinogen detoxification (Chaudhary et al. 2014). Broccoli cultivation has just lately started to take off in Egypt, which ranks 15th globally with a total output of 130,000 tonnes (Abou El-Magd and Zaki 2017). Several Brassica plant species are resistant to the harmful effects of HMs (Kaur et al. 2017), but according to Farooq et al. (2016), tolerance levels varied noticeably among different Brassica species variations.

Thus, in this study, it was vital to examine some physiological and biochemical reactions of broccoli and how closely these reactions relate to the mechanisms of As tolerance at various As levels. To our knowledge, no studies have been released about the effects of HA and MLE on broccoli’s ability to withstand As stress. There are also no experimental data on the effects of MLE on stress parameters under As conditions, but HA is recognized as an effective biostimulant to counteract As stress (Wang et al. 2021). This experiment used HA and MLE to describe and contrast the elimination of the detrimental effects of As on broccoli plants. To clarify the mechanism by which HA and MLE induce tolerance to As stress, this study focused on the roles of HA and MLE in protecting broccoli from various concentrations of As. The procedures were demonstrated via metabonomic study. The current study demonstrates how HA and MLE influence the toxic effects of As and main metabolic pathways and then offers insight into how HA and MLE control HM toxicity in plants.

2 Materials and Methods

2.1 Experimental Site

The broccoli vegetable (Brassica oleracea cultivar Italica) was planted in the greenhouse of the Botany and Microbiology Department at Assiut University (27°11′00″N 31°10′00″E) under natural conditions, from February to April, with day/night temperature of 21.7–31.4/6.3–14.5 °C and relative humidity of 42–28%.

2.2 Soil Characteristics

The air-dried utilized soil was sieved using a 2-mm sieve. The soil’s texture was loam (made up of 33.7% silt, 20.3% clay, and 46% sand), and its components included 1.76% organic matter, 183 mg kg−1 HCO3−, 360 mg kg−1 Ca2+, 144 mg kg−1 Mg2+, 37.8 mg kg−1 K+, 8.75 mg kg−1 available P, and 28.75 mg kg−1 available N with pH of 7.25 (Farghaly et al. 2022).

2.3 Humic Acid

Humic acid sodium salt was obtained from Sigma-Aldrich, Co., St. Louis, MO, USA. HA solution was freshly prepared by dissolving 0.1 g HA in 100 mL distilled water, and the pH was adjusted to 5.9 by HCl.

2.4 Moringa Leaf Extract

Young and disease-free Moringa oleifera leaves were collected from the New Valley Governorate, Egypt, rinsed with distilled water, air-dried at room conditions, and ground into a fine powder. The ground leaves (100 g) were mixed with 200 mL of absolute ethyl alcohol, left in a shaker for 4 h, and then filtered twice through the Whatman No. 1 filter paper (Rady et al. 2013). The mixture was evaporated with a rotary evaporator to evaporate the ethanol and dissolved in distilled water. The pH was adjusted to 6 by KOH, completed to 400 mL with distilled water, and then, the extract was sterilized using a 0.2-µm membrane filter.

2.5 Fourier Transform Infrared Spectroscopy

To detect the functional groups of HA and MLE, we employed FTIR (Nicolet IS 10 FTIR) in the Chemistry Department. Semi-transparent homogeneous tablets were prepared by a tablet-making machine using powder samples (about 100 μg) mixed with KBr (1:100 p/p). Spectra absorbance (400–4000 cm−1) was measured compared to a blank KBr pellet, and the resolution was 4 cm−1. Comparing the spectroscopic data with the references allowed us to identify the functional groups of the samples.

2.6 Plant Growth Conditions

The Department of Vegetables, Faculty of Agriculture, Assiut University kindly provided the broccoli seeds. Sterilized seeds were soaked in distilled water (control), the HA solution, and MLE at aeration conditions and 25 °C for 8 h. Ten soaked seeds were planted in a pot filled with 2.5 kg of soil and sand (2:1, v/v) and maintained at 100% field capacity by irrigation with tap water. Thirty days after planting, seedlings were thinned to three healthy plants per pot in preparation for beginning treatments. Plants were exposed to disodium arsenate heptahydrate (Na2HAsO4. 7H2O; AsV) in four concentrations (0, 0.75, 1.5, and 3 mM) throughout three doses. The AsV form predominates under aerobic circumstances (Wu et al. 2017). A simple experiment was conducted in a completely randomized block with four biological replicates and three plants in each pot. Three groups of pots were created. The first group, plants grown from seeds presoaked in water, was As only in a final concentration of 0 (control), 0.75 (As1), 1.5 (As2), and 3 (As3) mM (chosen after a preliminary examination). The HA group, plants grown from seeds presoaked in HA solution (0.1%), was combined with As levels (0 + HA, 0.75 + HA, 1.5 + HA, and 3 + HA). The MLE group, plants grown from seeds presoaked in MLE solution (25%), was combined with As levels (0 + MLE, 0.75 + MLE, 1.5 + MLE, and 3 + MLE). Sixty days after planting (30 days after As exposure), plants were harvested and fractionated into shoots and roots. The roots were only quickly rinsed with distilled water to prevent washing away of recently acquired As and gently blotted with filter paper. Shoots and roots were swiftly weighed separately to determine the fresh weight (FW), frozen in liquid nitrogen immediately, and kept at − 80 °C for future analysis. Other shoots and roots were oven-dried at 70 °C for 48 h to estimate the dry weight (DW).

2.7 Tolerance Index

The TI was calculated as the dry biomass ratio of shoot or root between the treated plant and the control and computed as a percentage (Farghaly et al. 2021).

TI (%) = DW of treated shoot or root/DW of control × 100.

2.8 Hydrogen Peroxide

The H2O2 content was measured spectrophotometrically, according to the Velikova et al. (2000) method. The sample was homogenized with 5 mL trichloroacetic acid and centrifuged at 12,000 rpm for 15 min. The supernatant was mixed with 2 mL of potassium phosphate buffer (PPB; 10 mM, pH 7.8) and 1 mL of KI (1 M) and left for 20 min. The absorbance of the sample was estimated at 390 nm using a spectrophotometer. The concentration was determined using the H2O2 standard curve and expressed as milligrams per gram FW.

2.9 Proline

Proline concentration was estimated using the acid-ninhydrin technique (Bates et al. 1973). The sample was homogenized in 3 mL of 3% sulfosalicylic acid and centrifuged at 10,000 rpm for 10 min. One milliliter of the aliquot was combined with 2 mL of acid-ninhydrin, and 1 mL of glacial acetic acid for 1 h at 100 °C. Following the termination reaction in the ice bath, 4 mL of toluene was added to the mixture and forcefully stirred. Toluene was used as a blank to measure absorbance at 520 nm, and a standard curve was used to determine the proline concentration and expressed as milligrams per gram FW.

2.10 Free and Bound Phenolic Compounds

The content of free and bound phenolic compounds was estimated using the Folin-phenol Ciocalteu’s reagent technique (Kofalvi and Nassuth 1995). The sample was extracted in 6 mL of 50% methanol for 90 min at 80 °C and centrifuged at 14,000 rpm for 15 min, and then, free phenolics were calculated. Pellets were combined with 2 mL NaOH for 24 h at room temperature, neutralized with 0.5 mL of HCl, and centrifuged at 14,000 rpm for 15 min. Methanol or NaOH extract was diluted with water to 1 mL and mixed with 2.5 mL of Na2CO3 and 0.5 mL of 2 N Folin-Ciocalteu phenol reagent for 20 min at room temperature. The absorbance of the sample was estimated at 725 nm using a spectrophotometer, and then, the phenolic content was calculated as milligram per gram FW from the gallic acid calibration curve.

2.11 Thiol Compounds

2.11.1 CYSTEINE

Cys concentration was estimated using the ninhydrin-acid reagent technique (Gaitonde 1967). Leaves (0.3 g) were homogenized in 3 mL of 5% cold perchloric acid and centrifuged at 13,000 rpm for 20 min at 4 °C. Two milliliters of supernatant were mixed with 1 mL of acid-ninhydrin reagent and kept in boiling water for 10 min, and then, the mixture was cooled rapidly. The absorbance of the sample was estimated at 560 nm using a spectrophotometer, and then Cys content was calculated as milligrams per gram FW from the Cys calibration curve.

2.11.2 Reduced Glutathione

GSH was estimated using the method of Anderson et al. (1985). Leaves (0.3 g) were ground in liquid nitrogen and mixed with 3 mL of sulfosalicylic acid, and then, the mixture was centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant (0.5 mL) was combined with 0.5 mL of reaction buffer (PPB (pH 7.0), ethylenediaminetetraacetic acid, disodium salt (Na2-EDTA), and 5–5′-dithiobis-2-nitrobenzoic acid (DTNB)) for 5 min. The absorbance of the sample was estimated at 412 nm using a spectrophotometer, and then, GSH content was calculated as milligrams per gram FW from the GSH calibration curve.

2.11.3 Non-protein Thiols

NPT content was estimated as described by Israr et al. (2006). As previously prepared, 0.5 mL of the supernatant was mixed with 0.5 mL of reaction buffer and kept for 10 min at room temperature. The absorbance of the sample was estimated at 412 nm using a spectrophotometer, and then, NPT content was calculated as milligrams per gram FW from the Cys calibration curve.

2.12 Enzyme Extraction

Leaves (0.5 g) were pulverized in liquid nitrogen and homogenized in 5 mL of 100 mM PPB (pH 7.8) containing 0.1 mM Na2-EDTA and 0.1 g polyvinylpyrrolidone. The mixture was centrifuged at 18,000 rpm for 10 min at 4 °C. The protein content of the supernatant was measured using bovine serum albumin as the standard curve (Lowry et al. 1951). The absorbance difference in wavelength per milligram of protein per minute was applied to calculate the enzyme activity.

2.12.1 Lipoxygenase

LOX (EC 1.13.11.12) activity was estimated as described by Minguez-Mosquera et al. (1993). Linoleic acid (0.035 mL) was mixed with 5 mL of distilled water containing 0.05 mL Tween-20 and kept at pH 9.0 by adding 0.2 M NaOH until complete transparency, and then, the pH was adjusted to 6.5 by HCl (0.2 M). The enzyme extract was mixed with the prepared solution, increased volume to 100 mL by 0.1 M PPB, and then absorbance was measured at 234 nm.

2.12.2 Catalase

CAT (EC 1.11.1.6) activity was estimated using Aebi (1984) method. The reaction medium (3 mL) contained PPB (pH 7), 100 µL H2O2, and 20 µL of the enzyme extract, and then the absorbance was estimated at 240 nm.

2.12.3 Peroxidase

POD (EC 1.11.1.7) activity was estimated using Zaharieva et al. (1999) method. The reaction medium (3 mL) contained PPB (pH 7), 6.5 mM of H2O2, 1.5 mM of guaiacol, and 100 μL of the enzyme extract, and then the absorbance was measured at 470 nm.

2.13 Statistical Analysis

In the treatments, there were three plants per pot and four pots per treatment. The results represent an average of three technical calculations across four biological replicates (± SD). We applied SPSS (version 22) to analyze the data. A one-way ANOVA was conducted following the post hoc Tukey multiple-range test (at P ≤ 0.05) to compare the means of parameters at each level of As treatment with/without HA or MLE. To evaluate the magnitude effects of each component or the interaction between each treatment and As, eta squared (ɳ2) was calculated as ɳ2 = SSEffect/SSTotal. Pearson’s correlation was used to determine the relationship between the mean values of various parameters under As, HA, or MLE treatments. Using Excel t-test, asterisks denote statistically significant differences between treatments with/without As (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ns = insignificant).

3 Results

3.1 Fourier Transform Infrared Spectroscopy

The functional groups characterizing HA and MLE were illustrated using FTIR spectroscopy (Fig. 1). The distinctive absorption peaks for the HA spectra emerged in three regions: the broad at 3410.72 cm−1 (stretch O–H band and phenolic compounds vibration) and two sharp at 1590.76 cm−1 (stretch C = O band and lignin vibration), and at 1380.75 cm−1 (CH2 and CH3 and polysaccharide vibration). Regarding MLE, the distinctive absorption peaks for the MLE spectra appeared in five areas: the broad at 3380.73 cm−1, and four sharp at 2922.98 cm−1 (stretch CH2 band and lipids vibration), at 1654.39 cm−1 (C = O and amide I vibration), 1420.01 cm−1 (O–H and cell wall polysaccharide vibration), and 1060.25 cm−1 (C-O and cell wall polysaccharide vibration).

Fig. 1
figure 1

FTIR spectra of the biostimulant compounds, where (A) is for humic acid and (B) is for moringa leaf

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3.2 Tolerance Index

Broccoli TI was measured to estimate the extent of As-induced harm to plant growth. The TI, which assessed the DW gain of the studied plants as a percentage compared to As-unstressed plants (controls), has been altered by As (Fig. 2A, B). The different As levels significantly reduced shoot and root biomass whence of TI compared to As-unstressed plants, and the highest reduction was 51.07% for shoots and 73.76% for roots at As3 (3 mM), respectively. Treatments of As-stressed plants with HA or MLE considerably enhanced the TI more than As-stressed plants alone. Compared to As-stressed plants, HA application increased shoot TI by 12.75%, while root TI was increased by 23.66% at As3, respectively. Regarding MLE treatment, compared to As-stressed plants, the increase was only 14.90% for shoots and 52.51% for roots at As3, respectively. Compared to the absolute control, plants treated with HA or MLE alone had no noticeable effect on TI. The two-way ANOVA analysis showed considerable variations between levels of every prime factor. MLE + As provided a medium influence (ɳ2 = 0.021 for shoots and ɳ2 = 0.017 for roots) in all treatments, according to the two-way analysis.

Fig. 2
figure 2

Tolerance index of shoots (A) and roots (B) of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant)

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3.3 Hydrogen Peroxide

According to Fig. 3A, B, As stress raised the H2O2 content in leaves and roots at all As levels, and the maximum stimulation at the As3 (3 mM) level was 89.61% for leaves and 223.27% for roots, respectively, compared to the control. However, HA and MLE treatments dramatically reduced the H2O2 content in As-treated leaves and roots. The study found at the As3 level in leaves that HA treatment reduced H2O2 accumulation by 29.92% and MLE by 48.78%; however, in the roots, HA reduced its accumulation by 42.07%, and MLE decreased its content by 63.70%, respectively, compared to As-stressed plants. Under normal conditions, neither the HA nor the MLE treatments appreciably decreased the H2O2 content compared with the absolute control. According to ƞ2 values, MLE + As treatment had the highest size effect on H2O2 content in leaves and roots (0.098 and 0.233, respectively). Results revealed that H2O2 content and TI of shoots and roots had strong negative correlations under normal conditions (− 0.951**, − 0.737**, respectively). The association between the H2O2 content and TI under As + HA treatment was modest (− 0.422) for leaves, while it was significantly negative (− 0.523*) for roots. Contrarily, with the As + MLE treatment, the relationship between the H2O2 content and TI was considerably negative (− 0.701**) for the leaves and modest (− 0.311) for the roots.

Fig. 3
figure 3

Hydrogen peroxide (H2O2) of leaves (A) and roots (B) of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant)

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3.4 Proline

To investigate the effect of As on the osmoprotectant, proline content was measured in the leaves and roots of broccoli plants subjected to As with/without HA or MLE (Fig. 4A, B). As-treated plants had higher proline content, and the maximum stimulation at the As3 (3 mM) level was 118.96% for leaves and 106.97% for roots, respectively, compared to the control. HA or MLE treatments reduced the proline content in leaves and roots compared to As-stressed plants. Compared to As-stressed plants at As3, proline concentration was reduced in HA + As-treated leaves and roots by 55.97% and 39.05% and by 40.49% and 25.90% in MLE + As-treated leaves and roots, respectively. Plants treated with HA or MLE alone did not show appreciable changes in the proline content of their leaves and roots. The values of ƞ2 showed that HA + As treatment had the highest size influence on the proline content in the leaves and roots (0.135 and 0.074, respectively). The findings also showed that proline content and TI of leaves and roots exhibited strong negative relationships when grown in As with HA, MLE, or neither.

Fig. 4
figure 4

Proline of leaves (A) and roots (B) of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant)

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3.5 Phenolic Compounds

Free and bound phenolics were evaluated to test whether As at different concentrations with/without HA or MLE affected the phenolic production of broccoli leaves (Fig. 5A, B). Results indicated that free phenolic concentrations in leaves were considerably stimulated by As, with the highest stimulation occurring at the highest As concentration (48.38% over As-unstressed leaves) (Fig. 5A). Supplementations of As combined with HA or MLE significantly/non-significantly reduced free phenolic content in leaves. In addition, no significant decrease in free phenolic content was recorded in plants treated with HA or MLE alone compared with the absolute control. The two-way analysis manifested that the MLE application had the highest size effect on free phenolics in the leaves (ƞ2 = 0.065). Furthermore, the data demonstrated that free phenolics and TI of shoots had significant negative correlations with either As + HA or As + MLE (− 0.908** or − 0.862**, respectively) or without HA or MLE (control, − 0.843**).

Fig. 5
figure 5

Free (A) and bound (B) phenolic compounds of leaves of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant)

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Regarding bound phenolics, the increase of As in the nutritional medium led to a decrease in levels of bound phenolics in broccoli leaves (Fig. 5B). Under the highest As concentration, there was a 46.74% reduction in the bound phenolic content compared to the control. However, the decrease in bound phenolics was not alleviated by either HA or MLE treatments. Under non-As toxicity conditions, HA or MLE treatments did not considerably change levels of bound phenolics in leaves compared to the absolute control. The MLE + As treatment had the highest size effect on the bound phenolic content in the leaves, based on the ƞ2 value (0.032). Furthermore, the results showed that As-treated plants with/without HA, MLE, the bound phenolic content, and TI of shoots exhibited strong positive associations.

3.6 Thiol Compounds

It was intriguing to find out whether HA or MLE would increase the resistance of broccoli plants to As toxicity and whether any of these effects would be connected to thiol substances, such as Cys, GSH, and NPTs. Therefore, the thiol compound contents in broccoli were estimated to explore the influences of HA or MEL on thiol compounds in As-treated broccoli.

3.6.1 Cysteine

According to the findings in Fig. 6A, As treatments reduced the Cys content in broccoli leaves, with the maximum reduction at As3 (3 mM) being 68.68% less than the absolute control. Applications of HA or MLE to As-stressed plants reduced the detrimental effect of As on the Cys content of leaves, and an increase of 198.06% for HA and 113.37% for MLE at the As3 level, respectively, was observed compared to As-stressed plants. Plants treated with HA or MLE alone showed a significant increase in Cys content compared to the absolute control. The two-way analysis showed that the highest size effect on Cys content in the leaves was observed in the MLE + As treatment (ƞ2 = 0.064). Additionally, As treatments with/without HA showed significant positive relationships between Cys content and shoots TI (0.646** and 0.872**, respectively), while these correlations were non-significant in the case of MLE treatments.

Fig. 6
figure 6

Cysteine (A), glutathione (B), and non-protein thiols (C) of leaves of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant)

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3.6.2 Glutathione

The toxicity of As had inhibitory effects on leaf GSH content at high As levels (As2 and As3); however, As1 failed to induce the same response compared with the control (Fig. 6B). However, treating HA or MLE with As-stressed leaves considerably boosted GSH content, with the highest As level recording the highest GSH levels compared to As-stressed leaves solely (304.78% and 137.92%, respectively). Regarding normal conditions, HA or MLE alone reduced GSH content in leaves relative to the absolute control. Additionally, HA with As treatment showed the highest size effects on GSH content in leaves (ƞ2 = 0.365). Also, As + HA and As + MLE demonstrated substantial negative correlations (− 0.751** and − 0.856**, respectively) between GSH content and shoots TI, while these correlations were considerably positive (control, 0.820**) in the absence of HA or MLE.

3.6.3 Non-protein Thiols

The data in Fig. 6C showed that As treatments had considerable stimulating effects on the NPT content in leaves of 35.65%, 48.98%, and 71.16% at As1, As2, and As3, respectively, over the control. When treated with As, HA substantially increased the NPT content in leaves compared to those treated with As alone. For instance, HA treatment increased the NPT content and was higher than the corresponding As1, As2, and As3 treatments alone by 49.45%, 69.40%, and 67.2%, respectively. Nevertheless, MLE treatment combined with As did not significantly affect the NPT content in leaves compared to As-stressed plants alone. No significant change was observed in the NPT content by application of HA or MLE alone to plants. Further, the two-way analysis manifested that the NPT content in leaves was most significantly affected by As + HA treatment (ƞ2 = 0.120). In addition, As treatments with/without HA or MLE revealed significant negative associations (− 0.972**, − 0.926**, and − 0.926**, respectively) between the NPT concentration and shoot TI.

3.7 Enzymes

3.7.1 Lipoxygenase

The increased As levels stimulated LOX activity in leaves (Fig. 7A). The maximum activation of LOX activity was recorded at the highest As (3 mM) level by 176.06% over the control. Applications of HA or MLE to As-stressed plants significantly reduced LOX activity in leaves by 16.41%, 19.00%, and 29.69% for HA and 8.52%, 7.53%, and 15.00% for MLE, respectively, which is less than that found in As1, As2, and As3-stressed leaves alone. HA applied to plants alone resulted in a considerable decrease in LOX activity, while MLE treatment did not significantly differ from the absolute control. Based on the ƞ2 values, HA and HA + As treatments had the highest size influence on LOX activity in the leaves (0.102 and 0.0421, respectively). Also, the findings illustrated that LOX activity and TI of shoots had strong negative correlations under As with HA or MLE (− 0.944** or − 0.939**, respectively) or without HA or MLE (− 0.876**).

Fig. 7
figure 7

Lipoxygenase (LOX; A), catalase (CAT; B), and peroxidase (POD; C) of leaves of broccoli plants under the influence of different concentrations of arsenate and humic acid (HA) or moringa leaf extract (MLE) for 60 days. The different letters, capital for arsenate treatments and small for HA and small with $ for MLE treatment, indicate significance, and asterisks indicate significant differences between control treatments and HA or MLE treatment (* P < 0.05; ** P < 0.01; *** P < 0.001; ns = non-significant).

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3.7.2 Antioxidant Enzymes

CAT and POD are the two most significant enzymes for regulating the intracellular level of H2O2. It will be interesting to know whether HA or MLE would increase plant resistance to As and whether these effects would be related to essential antioxidant enzymes such as CAT and POD.

Catalase and Peroxidase

Based on the data in Fig. 7B, As treatments considerably boosted CAT activity in leaves by 31.74%, 321.45%, and 900.15%, respectively, at As1, As2, and As3 compared to the control. Applications of HA or MLE along with As reduced the positive effects of As on CAT activity in leaves, showing a decrease by 11.10%, 37.62% for HA, and 42.34%, 33.74% for MLE, respectively, at As2 and As3, compared to the As-stressed plants. However, co-application of HA or MLE with As1 (0.75 mM) significantly/non-significantly enhanced CAT activity in leaves compared to As-stressed plants. Supplementations of HA or MLE alone to control plants caused a significant increase in leaf CAT activity compared to the absolute control. Furthermore, CAT activity in the leaves was significantly affected by As + AH or As + MLE treatments in the two-way analysis (ƞ2 = 0.067 and 0.062, respectively). In addition, results indicated a strong negative correlation between CAT activity and TI for plants treated with As + HA or As + MLE or without HA or MLE (− 0.773**, − 0.819**, and − 0.702**, respectively).

Regarding POD activity, As stress with/without HA or MLE had a favorable impact on leaf POD activity (Fig. 7C). In comparison with the control, the activity of POD was enhanced in leaves under As stress by 49.51%, 109.89%, and 139.69% at As1, As2, and As3, respectively. Co-application of HA or MLE with As stress showed a significant reduction in POD activity compared to As-stressed leaves alone, with decreases of 19.17%, 20.16% for HA and 13.76%, 10.96% for MLE at As2 and As3, respectively. Nonetheless, treatments of HA or MLE combined with As did not show a significant increase in POD activity in leaves at As1 (0.75 mM) compared to As-stressed leaves. Under normal conditions, plants treated with HA or MLE alone did not significantly alter POD activity compared to the absolute control. Moreover, the outcomes showed a high negative connection (− 0.951**, − 0.952**, and − 0.915**, respectively) between POD activity and TI of shoots of plants treated with As + HA or MLE and without HA or MLE.

4 Discussion

As was discovered to contribute to the overproduction of ROS induced by (a)biotic stress under these stressful circumstances, maintaining metabolic activities is necessary to prevent oxidative damage. This requires a balance between the creation and breakdown of ROS. Under stressful situations, plants devote most of their energy to strengthening their defense mechanisms rather than focusing on growth and development (Howladar 2014). It was found that the antioxidant system regulates the production of ROS in plant cells (Schützendübel and Polle 2002). Consequently, the level of the antioxidant system was anticipated to rise with the exposure of broccoli plants to As stress. The intriguing finding from this study is that applying HA or MLE to broccoli plants increased the antioxidant system's effectiveness in reducing ROS stress, whether As stress was present or not. There has been no study on the role of MLE in increasing plant tolerance to As stress, although the protective effects of HA against As stress have been explored in a few plant species, including lettuce (Wang et al. 2021).

Our results showed that the FTIR spectrum of HA spectra emerged in three regions: phenolic compounds, lignin, and polysaccharides (Türker-Kaya and Huck 2017). Fakour and Lin (2014) reported that the FTIR spectra of the analyzed humic compounds showed the presence of carboxyl, ketone, phenol, and hydroxyl functional groups. Also, Ephraim and Allard (1997) illustrated that the high affinity of humic compounds for trace elements in the aquatic environment is attributed to the existence of these functional groups. HAs can exchange ions, allowing them to chelate metal ions and use them as filters to capture HMs or remove environmental contamination from these metals in handling waste (Vašková et al. 2023). Besides heterogeneous dissociation, reactions between ROS and the functional groups, phenol, carboxyl, and polyphenol hydroxyl can also release a hydrogen radical, contributing to the antioxidant properties of these compounds (Vašková et al. 2023). Concerning MLE, FTIR analysis presented four areas: phenolic compounds, lipids, amide I, and cell wall polysaccharides (Türker-Kaya and Huck 2017), and these groups could chelate metallic ions. The physiologically varied bonds in these functional groups consist mainly of negative charges that can interact with the positive charge of the HM ion through an ion exchange process. According to Ravikumar and Udayakumar (2021), nearly all the functional groups in the M. oleifera gum complex are efficient groups for eliminating HMs. The authors mentioned that a metal atom may be substituted for a hydrogen atom in functional groups that include hydrogen, such as N–H and O–H.

The TI measures how much growth of the tested plants grown on media containing As is inhibited compared to the control plants. The TI has been used in numerous studies to assess plants’ susceptibility to various stressors, including As (Zvobgo et al. 2018). According to our results, As stress reduced plant growth, as indicated by lower shoot and root TI measurements (Zvobgo et al. 2018). As stress also affected the TI of roots more than shoots. In a review by Martínez-Castillo et al. (2022), membrane oxidation and plant metabolic disturbance are the main reasons for the effect of As on plant growth. According to Ullrich-Eberius et al. (1989), As can interact with the sulfhydryl groups of enzymes and tissue proteins, limiting cellular activity and even leading to plant and cell death. Applications of HA or MLE improved the TI in As-stressed plants, with the effect of MLE being greater than HA on the TI of shoots and roots. These results suggested that MLE and HA were crucial in reducing the impact of As on plant growth due to the presence of functional groups that may chelate As ions that produce oxidative stress. Improved TI of shoots and roots might result from the abundance of phytohormones and antioxidants in MLE. MLE is a rich source of certain antioxidants, soluble sugars, minerals, and amino acids, which may be why bean plants exposed to Cd, NaCl, or Cd + NaCl under stress grew in a healthy metabolic state when treated with MLE (Howladar 2014). Concerning HA, high TI of shoots and roots by HA suggested a reduced uptake of As. The humic compounds in humus are organic polyelectrolytes that are naturally occurring and stabilize soil-based organic materials. HA improves the physio-chemical properties of the soil, which enhances plant development and production directly or indirectly. Several authors have announced the capability of humate compounds to promote the growth of various plant species growing in different stressful environments (Sergiev et al. 2013). HA increased the lettuce growth by decreasing the transport of As from root to shoot, lowering the load on leaves and plastids (Wang et al. 2021).

There is significant evidence that plants exposed to inorganic As create ROS (Li et al. 2021) even though As is not a redox metal. As stress causes concurrent oxidative stress primarily by inhibiting electron transport during respiration and photosynthesis, which causes a significant buildup of ROS (Kaya et al. 2020). The presence of more H2O2 is a well-known sign of oxidative damage. In this study, As treatments raised H2O2 content in broccoli leaves and roots, with leaves having higher levels than roots. These results suggest that ROS, reflected by higher levels of H2O2, may contribute to the disturbance of the electron transport chain in plastids and mitochondria during As stress. Additionally, the findings showed that during As stress, leaves had relatively greater H2O2 levels than roots, indicating that plastids created more ROS. In line with this finding, the increased H2O2 level of plants during As toxicity was earlier found in maize plants by Kaya et al. (2020). Nevertheless, HA or MLE applications significantly decreased As-induced H2O2 accumulation in leaves and roots, confirming the critical role of these molecules in reducing As-stress-induced oxidative stress. Moreover, HA and MLE treatments successfully reduced the H2O2 content of As-stressed plants, and MLE had a higher effect than HA, especially in the roots. The role of HA and MLE in mitigating stress is probably due to their role in scavenging ROS and reducing As buildup. Similar to how it reduced As-induced oxidative stress in lettuce leaves and roots, HA also promoted the growth of lettuce plants by decreasing As-induced phytotoxicity (Wang et al. 2021). In addition, MLE treatment of bean plants under Cd stress reduced lipid peroxidation (Howladar 2014).

Proline has been demonstrated to shield plant cells from toxic HMs by creating non-toxic complexes with harmful metals (Ahmad et al. 2021). In our study, the proline content of leaves and roots increased with increasing As supply, suggesting that proline production may help plants resist As toxicity. We prefer to hypothesize that proline rose with rising As and declining plant growth, which is consistent with the significant negative association between proline and TI of shoots and roots. According to our research, the plants with As-tresses may not have collected enough proline to completely undo the oxidative damage brought on by lipid peroxidation and high H2O2 production. Accordingly, Chandrakar et al. (2017) concluded that proline at a high concentration directly scavenges hydroxyl radicals and reduces As accumulation in Glycine max, protecting against As toxicity. Furthermore, proline is known to act as a ROS-eliminator, and, thus, a significant buildup of proline under As stress may indicate an osmotic change (Kaya et al. 2020). HA and MLE treatments controlled proline accumulation in As-stressed plants, suggesting that the beneficial effects of HA and MLE on As-stressed plants might be due to a reduction in the ROS rise brought on by As-induced stress. Additionally, HA treatment had a higher impact than MLE treatment in lowering the proline level in As-stressed leaves and roots, indicating that HA was more effective in lowering As uptake. In this respect, Yildiztugay et al. (2019) found that HA application significantly reduced the proline content in garden cress plants of Cd-stressed plants, and the authors attributed this finding to the presence of many carboxyl and hydroxyl groups which suppress ROS generation. Although MLE treatments can reduce HM stress (Amorim et al. 2023), no study in the literature shows that MLE treatments decrease proline levels in plants under metal stress.

Phenolic compounds are among the predominant secondary metabolites in plants. Additionally, they are among the antioxidants that eliminate excess ROS caused by stress. Phenolic compounds can be divided, into soluble and insoluble groups, based on their location and chemical makeup inside the plant (Reis Giada 2013). Free simple phenolics, glycosides (containing one or more sugar moieties), and low-molecular-weight phenolics that are not linked to the other essential elements of plant cells make up soluble phenolics. Few studies have been conducted on phenolics under As stress, according to Martínez-Castillo et al. (2022). According to the findings, As stress caused a considerable rise in free phenolics in As-stressed leaves, which may have caused oxidative damage. The strong negative correlations between TI and free phenolic content in leaves confirmed that plants might use their energy to continue growth in As-contaminated soils. Tolerance may involve a plant’s capacity to increase the production of As-chelating substances like phenolics, enabling the plant to withstand the oxidative stress brought on by As toxicity. Kofroňová et al. (2020) also discovered that As treatment probably enhanced total phenolic content, and the authors hypothesized that phenolics significantly contributed to the defense, particularly in tobacco Wisconsin genotype roots. Treatments with HA or MLE reduced the positive effects of As on free phenolic content, and this inhibition suggested mitigation of oxidative damage, as supported by a decrease in LOX activity and H2O2 buildup. Moreover, Alu'datt et al. (2019) found that in the Solanaceae and Oleaceae fruit families, free phenols have more antioxidant capabilities than bound phenols. No study available in the literature indicates that HA or MLE reduced free phenolic production in As-stressed plants.

Bound phenolic groups include phenolic acids, condensed tannins, and other compounds of low molecular weight (Reis Giada 2013). The bound phenolics are ester-linked to cell wall polysaccharides and undergo modification in response to stress. The current study indicated that As stress with/without HA or MLE had a deleterious effect on leaves-bound phenolic content. The strong positive link between TI and bound phenolic levels in leaves supports prior studies by Gupta and De (2017), who concluded that bound phenolics are connected to plant growth. In our study, the significant reduction in bound phenolics of plants treated with HA + As or MLE + As compared to As-stressed plants reveals the suggestion of Fan et al. (2006) that the gradual accumulation of bound phenolics is related to the suppression of wall expansion and root growth by water deficiency in maize. The effect of As with/without HA or MLE on phenolics requires further investigation.

One of the main steps in As detoxification in plants is the complexing of As by thiol compounds, followed by its sequestration in vacuoles (Sun et al. 2021). The thiol compounds of broccoli plants treated with As with/without HA or MLE may offer information for enhancing plant growth. Products from S metabolism assist plants in overcoming HM stress (Mostofa et al. 2015). There are limited investigations on the associations between As toxicity and thiols, despite numerous studies on thiols’ use as an antioxidant to reduce HM oxidative stress.

Cys, a key metabolite, is a S donor for other S-containing compounds (Zagorchev et al. 2013). According to several lines of evidence, thiols play essential roles in As tolerance and detoxification (Mishra et al. 2008). The current study demonstrated that As stress had a detrimental effect on the Cys content of leaves, indicating a high accumulation of As and toxicity to plant metabolism as seen by a reduction in plant growth. The significant positive associations between TI and Cys levels in shoots support the earlier reports of Schmoger et al. (2000), who concluded that thiol compounds detoxify As toxicity. A similar adverse effect of As toxicity on Cys content was previously reported in Brassica juncea variety TM-4 (Srivastava et al. 2009). The authors concluded that after 7 days but not after 15 days, TM-4 showed increases in metabolite levels and thiol-metabolizing enzyme activities. Furthermore, according to Gupta and Ahmad (2014), the lower Cys level in the As (V) treated-IR-64 rice variety may be due to the usage of a weaker defensive mechanism against this particular form of As. Additionally, Bashir et al. (2013) reported that the accumulation of free Cys might cause unintended S loss, which impairs the plant’s stress tolerance. HA and MLE treatments increased Cys content in As-stressed leaves, suggesting that these treatments may inhibit the uptake of As by plants. There is no evidence in the literature that HA and MLE treatments increased Cys levels in plants, although Cys is known to control excess ROS under different abiotic stress. Zhang et al. (2014) reported that cell wall–bound HA could occupy the As uptake site by creating a protective floc layer and reducing As uptake and toxicity. Furthermore, MLE treatment controlled Na and Cd accumulation in Phaseolus vulgaris plants, which may improve salinity and Cd tolerance, according to Howladar (2014). Without As stress, HA or MLE treatments were active in increasing Cys accumulation, suggesting that these treatments could improve S uptake and Cys production, which would help the plants resist the toxicity of HMs. Also, the foliar application of MLE significantly increased the content of N, K, P, and S in brinjal fruit (Hoque et al. 2020). The results of numerous studies that demonstrate HA’s capacity to improve soil nutrient availability and plant uptake have been compiled (Ampong et al. 2022). According to Bonner et al. (2005), Cys synthesis is essential for plants to produce inorganic S from the environment since it acts as the only metabolic sulfide donor for making methionine, GSH, iron-sulfur clusters, vitamin cofactors, phytochelatins, and many secondary metabolites. Thus, the effect of As with/without HA or MLE on Cys requires further investigation.

GSH is an essential part of the ascorbate-GSH cycle and a precursor of PCs, which are used to detoxify HMs by sequestering them in vacuoles (Noctor et al. 2012). In this study, the high As treatments reduced the GSH levels in broccoli leaves, indicating increased NPT synthesis. As the As content increased in leaves, so did the demand for PCs, which led to the usage of GSH and Cys in PC synthesis. Additionally, increased H2O2 levels in plants imply that GSH depletion may result in oxidative stress, and the strong positive correlation between GSH and TI may support this suggestion. Our findings are consistent with earlier studies, which found that an increase in NPTs was associated with a drop in GSH and Cys levels (Bashir et al. 2013; Shri et al. 2014). However, HA and MLE treatments promoted GSH accumulation in As-stressed leaves, indicating that plant resistance to As has improved. The treatments of HA or MLE decreased H2O2 content, alleviating As-induced stress and further increasing the plant TI. Yildiztugay et al. (2019) showed that HA addition to wheat plants under Cd stress increased GSH and GSH/GSSG ratios, indicating that higher GSH and GSH/GSSG ratios were favorably associated with plant capacity to withstand Cd-induced oxidative damages. The improvement of GSH content in As-stressed plants by MLE may be due to the active participation of GSH in the detoxification of ROS and the control of intracellular oxidative stress. HA or MLE applications without As stress reduced GSH in plants, indicating that these treatments could enhance S buildup and minimize GSH synthesizing. In this respect, Nikiforova et al. (2003) concluded that S availability limits plant GSH accumulation. Further research is necessary to clarify the role of applied HA or MLE during As stress in plants.

NPTs are more susceptible to cellular oxidative stress changes because they preserve protein function and structure by limiting protein thiol oxidation (Yang and Guan 2015). During the current investigation, As + HA or As + MLE enhanced the accumulation of NPTs, indicating As chelation. Since broccoli contains significant quantities of S, it may have a crucial role in detoxifying As. Our results are consistent with a previous study that reported that the efficient synthesis of NPTs plays a critical role in achieving As tolerance (Kumar et al. 2019). Increased NPT concentrations caused by HA or MLE in As-stressed plants show how crucial this antioxidant is for protecting plants from oxidative stress brought on by As. Moreover, HA treatment had a higher effect than MLE treatment on stimulating NPT levels in As-stressed plants, suggesting that HA was more effective in chelating As.

Toxicity altered the redox state and reflected in increased LOX activity in broccoli leaves. The strong negative correlations between LOX activity and TI indicated that As increases LOX activity, which leads to lipid oxidation. The elevated activity of LOX is a sign of significant lipid damage brought on by As-induced ROS buildup (Chandrakar et al. 2017). Lipids and proteins may be damaged by an excessive buildup of H2O2 (Liu et al. 2020), which could compromise their effectiveness (Palma et al. 2019). HA and MLE treatments markedly suppressed LOX activity in the As-stressed broccoli leaves. Increased antioxidant activity may have contributed to the reduction in LOX activity brought on by HA and MLE. Similarly, HA and MLE have been demonstrated to be successful in reducing stress-related lipid peroxidation in Zea mays and Phaseolus vulgaris (Latif and Mohamed 2016; Kaya et al. 2020).

Antioxidant enzymes keep cell ROS at safe and low levels; as a result, these levels are crucial markers of a plant’s ability to react to stress (Khan et al. 2009). The main enzymes responsible for breaking down H2O2 are CAT and POD. The increased CAT and POD activities under As toxicity were observed in leaves, while As + HA and As + MLE treatments reduced their actions compared to As alone. These results demonstrated that whereas As toxicity increased the content of ROS, it also sped up defense mechanisms at low concentrations since H2O2 was still high. Increased antioxidant enzyme activity may increase plants’ tolerance to HMs (Valko et al. 2005). As-treated rice plants showed a similar pattern (Khan and Gupta 2018). As + HA– and As + MLE–subjected plants had decreased CAT and POD activities compared to As treatment alone, which suggests that these plants may have enough other endogenous antioxidants to eliminate the low ROS concentration. Likewise, HA treatments reduced CAT and POD activities in As-treated lettuce roots (Wang et al. 2021), and HA structures may interact with H2O2 (García et al. 2014). The MLE application led to a decrease in CAT and POD activities, suggesting the possibility of forming chelating complexes with MLE in the nutrient medium or in the plant. Further research is necessary to clarify the role of applied MLE on antioxidant enzymatic activity during As stress in plants.

5 Conclusion

Arsenate toxicity negatively affected the physiology and development of broccoli plants. The results showed that arsenate toxicity reduced plant growth, bound phenols, cysteine, and glutathione. However, the toxicity enhanced oxidative damage in plants, which was evident in increased levels of hydrogen peroxide, proline, free phenols, non-protein thiols, lipoxygenase, catalase, and peroxidase activity. On the other hand, humic acid and moringa leaf extract treatments boosted antioxidant levels in plant leaves, decreasing the growth inhibition and oxidative damage caused by arsenate toxicity. This probably happened due to the improved synthesis of thiol compounds, which conceivably have capacities in reactive oxygen species removal and heavy metals chelation, driving extended resistance to arsenate toxicity (Fig. 8). The examination of Fourier transform infrared spectroscopy spectra of humic acid and moringa leaf extract showed that functional groups, including phenolics, lignin, polysaccharides, lipids, and amide I, were involved in the elimination of reactive oxygen species produced by arsenate effects, highlighting their appropriateness as low-cost and ecologically friendly materials. Therefore, we can say that humic acid and moringa leaf extract are helpful and secure treatments that can aid plants in overcoming the negative impacts of arsenate stress, even though more study is required.

Fig. 8
figure 8

A demonstration for relief of arsenate (As) stress in broccoli plants by humic acid (HA) or moringa leaf extract (MLE). Here, As decreased the tolerance index (TI), bound phenolics, and thiol compounds while increasing H2O2, proline, free phenolics, LOX, CAT, and POD activity. HA or MLE application to As-stressed plants decreased H2O2, proline, phenolics, LOX, CAT, and POD while increasing plant growth (TI) and thiol compounds

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