The Eliciting Effect of Aqueous Extracts from Ascophyllum nodosum Algae on the Cultivation of Arugula (Eruca sativa Mill.) Microgreens

Abstract

This study showed the eliciting effect of aqueous extracts from Ascophyllum nodosum on the development of Eruca sativa microgreens in a quartz-based substrate. There is no information in the literature on the impact of its use on the quality, bioactive compound content, and nutritional value of arugula microgreens. Assays that have been made include the following: total phenolic content (TPC), total flavonoid content (TFV), enzymes such as phenylalanine ammonia (PAL) and polyphenol oxidase (PPO), and enzymes involved in the scavenging of reactive oxygen species such as catalase (CAT) and superoxide dismutase (SOD). The antioxidant activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) was also evaluated. The total phenolic content of arugula microgreens increased significantly after an application of aqueous extracts of A. nodosum to the substrate. The greatest increase in flavonoid content (89%) and antioxidant activity against DPPH (82%) was observed at a concentration of 2.5%. The highest increase in activity of CAT (68.2%), SOD (25%), PAL (13-fold), and PPO (84.2%) was observed with the application of 5% extract. In conclusion, the use of Ascophyllum nodosum algae affects TPC and TFV, antioxidant activity, PAL, PPO, SOD, and CAT in the microgreens studied. By prioritising organic and environmentally friendly extracts, growers can contribute to a more sustainable and healthier food system, especially in the case of the production of Eruca sativa microgreens.

1. Introduction

Microgreens are a relatively new class of edible vegetables that are harvested when the expanded cotyledons are fully developed before the true leaves emerge. They are young and tender leafy greens that come in a wide range of colours, textures, and flavours. These foods are becoming increasingly popular due to their higher concentration of bioactive compounds such as vitamins, minerals, and antioxidant activity compared to mature vegetables [1].

The microgreens of Eruca sativa (Brassicaceae family) are renowned for their nutritional and medicinal properties.

Among all the microgreens produced, the Brassicaceae stand out because of the extensive evidence of their beneficial effects on human health attributed to phenolic compounds, vitamins, and, in particular, glucosinolates and their degradation products, isothiocyanates and indoles [2].

Arugula leaves and their constituents have recently captured the interest of scientific communities worldwide due to their potent bioactivity and health-promoting properties [3,4]. The crude extract of E. sativa leaves contains a wide range of phytochemicals including amino acids, vitamins, fatty acids, alkaloids, flavonoids, terpenoids, and phenolic compounds. Phenolic compounds are among major constituents of arugula leaves that contribute to its antioxidant properties [5,6]. Phytochemical studies of Eruca sativa extracts have also shown that quercetin, kaempferol, and isorhamnetin glycosides are the main flavonols in this plant, with kaempferol being the major constituent [7].

A diet rich in vegetables has been widely recognised in the literature to provide numerous health benefits. These benefits are mainly attributed to the increased intake of phenolic compounds with high antioxidant capacity [8]. It is important to note that the antioxidant properties of plant tissues can vary depending on the species used, and the processing of fresh leafy vegetables for food preparation can have different effects [5]. Thus, the inclusion of leafy greens in the diet can have a positive effect on health.

Sustainable agriculture is important in modern crop production because it promotes environmentally and human-health-friendly practices that reduce the use of chemicals in crop production. Practises such as the use of biostimulants or ‘green fertilisers’ enhance the physiological and metabolic responses of plants to biotic and abiotic stresses, reduce nutrient losses, and increase crop efficiency. Their use also reduces the dependence on chemical fertilisers and synthetic pesticides, increases crop resilience, and promotes soil health and fertility. Green chemistry represents a new paradigm in agriculture, serving as a driver for sustainable agriculture and providing an intelligent way to produce and use formulations that do not harm the environment [9].

Bio-fertilisation is a sustainable agricultural practise that involves the use of bio-fertilisers to increase the nutrient content of the soil and organic matter, resulting in higher productivity. Micro- and macroalgae are the right environmentally friendly bio-based products for non-polluting agricultural applications. The use of bio-fertilisers is undoubtedly the future of agriculture, where they are expected to reduce the use of chemical fertilisers. This type of fertiliser is safer for the soil and also facilitates the process of biodegradation carried out by microorganisms, leading to an increase in soil fertility in a safe way, without leaving chemical residues [10].

Arugula contains many desirable phenolic and flavonoid compounds and other antioxidants. Their levels can vary depending on the variety, growing conditions, processing techniques, and environmental aspects or fertilisers used to adjust the phenolic and flavonoid content and antioxidant activity of Eruca sativa [5]. Optimising these factors can increase the amount of bioactive compounds and improve the nutritional quality of arugula. One factor that can determine the nutritional value of young arugula leaves or seedlings is the use of seaweed extracts.

Some plant hormones have been found in algae, including auxin, cytokinin, gibberellin, ethylene, jasmonic acid, abscisic acid, brassinosteroids, salicylic acid, and strigolactones [11]. Seaweed also contains trace elements (iron, zinc, molybdenum, copper, manganese, nickel, cobalt), amino acids, and many vitamins [12]. Treatment with seaweed extracts is known to have a significant effect on the cellular metabolism of plants, facilitating the uptake of essential elements from the soil and improving plant resistance to pests, disease, and stress [13].

The brown alga Ascophyllum nodosum (L.) Le Jolis is a common source of plant growth stimulants. It is used to improve the regulation of physiological, biochemical, and molecular processes and to enhance plant resistance to stress [14].

A. nodosum is a common intertidal species found along the periphery of the North Atlantic Ocean, particularly along the northwest coast of Europe (from Svalbard to Portugal), including eastern Greenland. It is highly efficient at accumulating nutrients and minerals from the seawater, making its biomass a valuable resource for industry and agriculture due to the presence of many bioactive compounds. This species has several commercial uses, including as a food additive, fertiliser, soil conditioner, plant biostimulant, animal feed, and active ingredient in skin and hair care products [15]. The amount of bioactive constituents and metabolites present in algae extracts varies depending on their geographical origin, reproductive stage, and exposure to environmental and collective stressors [16], as well as the extraction methods used. Kumari et al. [14] extensively reviewed the chemical composition of aqueous Ascophyllum biomass extract. Biostimulants produced by this specific method have been found to possess phytohormone-like activities and have been shown to benefit plant growth and stress tolerance [17,18,19].

There is little information in the literature on the potential use of these valuable algae in the cultivation of microgreens. For example, a study of oat microgreens found that soaking in an Ascophyllum nodosum algae suspension affected their germination, metabolism, and chemical composition. Plants from seeds soaked in suspensions had higher levels of functional sugar and protein building groups. However, no differences in lipid content were found [20].

Knowing that the alga Ascophyllum nodosum is a valuable source of antioxidants, including phenolics, polysaccharides, and pigments which have shown great potential in plant cultivation [21], we aimed to investigate its possible applications in microgreen production.

For the above reasons, it follows that research investigating the effects of Ascophyllum nodosum algae extracts on the bioactive compounds, enzyme activity, and health-promoting properties of arugula microgreens is significant and important.

Antioxidants can be classified into two main categories: enzymatic (e.g., superoxide dismutase, catalase, and oxidase enzymes) and non-enzymatic antioxidants (e.g., total phenolic compounds, total flavonoids, and DPPH scavenging activity). Plants are a particularly important source of antioxidants for humans [22].

The aim of the experiment was to evaluate the effect of A. nodosum algae extracts on the bioactive compounds of arugula microgreens. The presence of total phenolic compounds and flavonoids was determined, as well as the activity of enzymes responsible for the synthesis of bioactive compounds and the scavenging of ROS in plants. The presented experiment aimed to determine whether the levels and activities of certain enzymes were dependent on the concentration of the extract used. Additionally, this study aimed to identify a dose that would enhance the health-promoting properties of microgreens.

To summarise, arugula is known to be a nutritious and valuable plant but research into optimising its health-promoting properties is limited. Determining the concentration of seaweed extract is of great importance in order to increase bioactive compounds and enzyme activity while ensuring that the quality of the product is not compromised. The aim of this research project is to provide evidence-based recommendations for the production of arugula microgreens with optimised nutritional and health benefits through the strategic use of Ascophyllum nodosum seaweed extracts. The findings have the potential to benefit human health by increasing the availability of nutrient-dense, bioactive-rich specialty crops.

2. Materials and Methods

Reagents and laboratory equipment used in the experiment are provided in the Supplementary Materials (S1).

2.1. Preparation of Extracts

All extracts were prepared from commercially available (Calaya, Złotów, Poland) raw materials—dried, powered Ascophyllum nodosum algae—by adapting typical biomass extraction methods [23]. The UAE (ultrasound-assisted extraction) technique was used to prepare the extracts under dual-extraction conditions, including 30 min, 30 °C, 600 W, and 40 kHz, using an 1100 W Digital PRO PS100A ultrasonic bath (CNCTech, Poland). The solvent used for extraction was demineralised water with a conductivity of 0.05 μs, and the ratio of raw material to extract w/w was 1:20. After each extraction process, the extract was decanted from the extracted raw material and filtered by gravity through filter funnels with filter paper with a grammage of 75 g m⁻² and medium porosity (Biospace, Poznań, Poland). The filtered extracts were combined and then centrifuged in a rotary centrifuge EBA 200 (Hettich, Tuttlingen, Germany) at 6000 rpm for 5 min. The starting extract prepared in this way was analysed (see Section 2.2 and Section 2.3) and used to prepare dilutions. Subsequently, the plants were irrigated with preparations in the following proportions: 25 mL of extract + 975 mL of tap water (W2), 50 mL of extract + 950 mL of tap water (W3), and 100 mL of extract + 900 mL of tap water (W4). The control (tap water only) is designated as W1. Since the density of the extract was approximately 1 g⋅cm⁻³, it can be approximated that the dilution of the extract was 2.5% (W2), 5% (W3), and 10% (W4).

2.2. Mineral Composition of the Extract

The composition of macro- and microelements in the extracts was determined using ICP-OES Thermo iCAP Dual 6500 (Schaumburg, IL, USA) according to the procedure described by Mroczek et al. [24]. The mineral composition of the extracts is presented in

Table 1. Mineral composition of extracts in mg⋅L⁻¹.

Macroelements	Dose	Ca	Mg	K	P	S	Na
	W1	1.33 ± 0.16	1.93 ± 0.11	5.75 ± 0.44	Trace	5.46 ± 0.05	6.59 ± 0.13
	W2	1.90 ± 0.10	2.76 ± 0.05	14.1 ± 0.10	0.24 ± 0.03	7.624 ± 0.06	10.79 ± 0.12
	W3	3.76 ± 0.05	5.78 ± 0.09	29.01 ± 0.28	0.187 ± 0.02	16.28 ± 0.13	23.913 ± 0.24
	W4	7.96 ± 0.15	11.74 ± 0.10	57.64 ± 0.36	1.833 ± 0.031	32.43 ± 0.185	48.14 ± 0.337
Microelements	Dose	Cu	Zn	Cr	Fe	Mn	Mo	Sr
	W1	0.113 ± 0.015	0.063 ± 0.08	-	0.018 ± 0.03	0.006 ± 0.0	0 ± 0.0	0.047 ± 0.003
	W2	0.149 ± 0.1	0.065 ± 0.04	0.003 ± 0.01	0.022 ± 0.04	0.012 ± 0.01	0 ± 0.0	0.045 ± 0.001
	W3	0.110 ± 0.2	0.063 ± 0.01	0.001 ± 0.0	0.074 ± 0.06	0.024 ± 0.01	trace	0.097 ± 0.001
	W4	0.081 ± 0.2	0.069 ± 0.01	0.004 ± 0.0	0.254 ± 0.03	0.047 ± 0.005	0.001 ± 0.0	0.196 ± 0.002

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2.3. Qualitative Analysis of the Extract

A qualitative analysis of the aqueous extract of Ascophyllum nodosum (base extract before preparation of the doses) was performed. Test procedures for phytochemical assays [25,26,27,28,29,30,31,32,33,34,35,36,37] are provided in Supplementary Material (S2), as well as images from the experiments performed. The results of the assays are presented in

Table 2. Qualitative analysis of the extract.

Test Name	Phytoche Mical Group Detected	Sample Name	Positive Test Result—Description	Interpretation	Result Obtained
foam/emulsion test	saponins	7A	Persistent foam/formation of an emulsion with oil	foaming, emulsifying, and clouding were found	+
FeCl3 test	tannins	7B	gallic tannins—navy blue catechol tannins—dark green	dark brown gelatinous precipitate	-
gelatin test	tannins/phenolic compounds	7C	precipitation	no chemical reaction	-
alkaline test (NaOH)	anthocyanins/flavonoids	7D	Anthocyanins—blue colour after addition of NaOH, turning yellow or orange after addition of HCl; Flavonoids—intense yellow colour after addition of NaOH, colour fades after addition of HCl	The addition of sodium hydroxide (NaOH) causes the solution to darken, while the addition of hydrochloric acid (HCl) causes it to lighten.	tannins− flavonoids+
lead acetate test	tannins/flavonoids	7E	white precipitate—tannins; yellow precipitate—flavonoids	yellow precipitate	tannins− flavonoids+
Shinod test	flavonoids	7F	red colour of the solution	yellowish colour + gelatinous precipitate	-
Mayer test	alkaloids	7G	cream-coloured precipitation/green solution	precipitate after adding HCl	-
Wagner test	alkaloids	7H	brown-red precipitate	no chemical reaction	-
Dragendorff test	alkaloids	7I	orange precipitate	precipitate after adding HCl	-
Keller–Killiani test	cardiac glycosides	7J	cardiac glycosides—red-brown ring; (steroid aglycone—part of the glycoside)	very subtle darkening at the junction on two phases	inconclusive
Liebermann–Burchard test	cardiac glycosides/steroids/terpenoids	7K	cardiac glycosides—green colour/ring (steroid aglycone—part of the glycoside); steroids—green colour/ring (steroid aglycone—part of the glycoside); terpenoids—red-purple colour	greenish ring	glycosides/steroidal aglycones+
Salkowski test	steroids/terpenoids	7L	steroids—red-brown ring (steroid aglycone—part of the glycoside);terpenoids—red-brown ring on the interface	brown ring	terpenoids+
Fehling test	reducing sugars/carbohydrates	7M	brick-red precipitation	blue-green solution	-
Biuret reaction	proteins and amino acids	7N	violet coloration	gelatinous precipitate/green tint	-
Xanthoproteic reaction	proteins and amino acids	7O	After adding HNO3, a white precipitate forms. After dissolving it in ammonia, it turns yellow.	after HNO3—no precipitate; after adding N3—the yellow one	-
Borntrager test	anthraquinones	7P	pink, purple, or violet colouring	brown-yellowish colouration with subtle pinkish tint	inconclusive
Molish test	reducing sugars/carbohydrates	7R	reddish-purple ring at the phase boundary	yellowish colouration with slight darkening at the junction of the phases	inconclusive

.

The prepared sample was also analysed by SPME (solid-phase microextraction) using a 100 µm polydimethylsiloxane (PDMS) fibre (Supelco Ltd., Bellefonte, PA, USA). Prior to analysis, this fibre was conditioned at 250 °C for 30 min in a gas chromatograph dispenser according to the manufacturer’s instructions. The tested material (heated to 45 °C) was placed in a 100 mL conical flask which was secured with aluminium foil. The fibre was transferred to the gas chromatograph injector (temp. 250 °C) where the analytes were thermally desorbed for 5 min. The composition of the compounds desorbed from the SPME fibre was investigated using a gas chromatograph (GC-MS, Varian 450GC compressed with 240 MS). Helium was used as a carrier gas at a flow rate of 1 mL/min. The temperature of the dispenser was 250 °C. The separation of the analytes was carried out using a 30 m × 0.25 mm capillary column with a moderately polar HP-5 (polysiloxane-methylphenyl) stationary phase and a layer thickness of 0.25 µm. The column oven temperature programme was as follows: start at −50 °C for a 5 min isotherm, and then set to a temperature gradient of 10 °C/min to 300 °C (5 min isotherm). Compounds found in the extracts were identified using NIST.08 and the Willey database. GC-MS analysis was carried out in duplicate.

The results of the analysis are presented in

Table 3. Chemical composition of headspace fractions of A. nodosum water extract.

No.	RT [min]	Peak Share in the Chromatogram [%]	Ordinary Substance Name	Systematic Substance Name	No CAS
1	4.75	1.17		1,1-Dimethylsilanediol	1066-42-8
2	7.90	12.44		5-Chloroindole	17422-32-1
3	9.80	3.75		Octenal	25447-69-2
4	10.91	3.27		Benzene, 1-chloro-3,5-bis(1,1-dimethylethyl)-2-(2-propenyloxy)-	55955-96-9
5	13.72	19.21		(E)-2-decenal	3913-81-3
6	14.19	4.81		Dodeca-2,4-dienal	13162-47-5
7	15.16	10.12		3-(bromomethyl)cyclohexene	34825-93-9
8	15.64	4.77		2-Cyclohexene-1-methanol, 2-methyl-a-(trichloromethyl)-	103659-46-7
9	16.58	6.43		2,6-di-tert-butylo-p-benzochinon	719-22-2
10	16.82	20.14	(E)-beta-ionone	4-(2,6,6-Trimethyl-1-cyclohexenyl)-3-buten-2-one	79-77-6
11	17.89	9.38		(E,Z,Z)-2,4,7-tridecatrienal	-
12	21.03	4.44		Diisobutyl phthalate	84-69-5
13
14
TOTAL

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2.4. Pot Experiment

The plants (Eruca sativa, TORAF, Kluczbork, Poland) were seeded and cultivated in a controlled pot experiment under optimal conditions of an air temperature of 20 °C and 80% humidity in a growth chamber. The growth chamber produced photosynthetically active radiation ranging from approximately 400 to 700 nm with an intensity of 800 µmol·m²/s⁻¹. The day/night photoperiod consisted of 10 h of light followed by 12 h of darkness. The experiment used a loose substrate primarily composed of quartz mineral. The substrate had a pH of 6.0 in H₂O and contained 9.39 mg of P₂O₅ per 100 g of soil and 20.8 mg of K₂O per 100 g of soil of bioavailable forms of P and K, respectively. The seeds used in the experiment were intended for sowing sprouts. The seeds were marked with the batch number 010685CINOFS and had the PL-EKO-07 organic farming certificate. These seeds were produced without the use of chemicals and mineral fertilisers in accordance with European Union standards. The dimensions of the pots used in the experiment were 20 × 15 × 4 cm, and 3 g (about 1000 seeds) were sown in them.

The plants were watered with a fresh control solution—tap water (W1)—and solutions of water containing labelled algae extracts—W2, W3, and W4. Each experimental object consisted of 20 pots. The pot experiment used a completely randomised design.

The irrigation occurred every 24 h (for a period of five days). The experiment was carried out for 6 days from the time of germination. All experimental objects were removed at the same time. The above-ground biomass (about 5 g from each pot) was subsequently harvested and analysed to investigate the impact of irrigating with solutions of Ascophyllum nodosum seaweed on the production of specific bioactive compounds within the plant material.

2.5. Analysis of Antioxidant Activity Assay in Microgreens

After appropriate sample preparation, described in detail in the Supplementary Materials (S3), the antioxidant activity (DPPH) was measured and the results are expressed as Trolox equivalents. Phenolic content is expressed as gallic acid equivalents [38]. Flavonoid content is expressed as quercetin equivalents (S3).

2.6. Determination of Enzymatic Activity

SOD activity was measured using a spectrophotometric method based on the inhibition of epinephrine autoxidation according to Piechowiak and Balawejder [34]. Catalase (CAT) activity was determined using ammonium metavanadate according to Hadwan and Ali [35] with minor modifications. PPO (polyphenol oxidase) activity was measured by a colourimetric method using pyrocatechol as the substrate [38]. The activities of the above enzymes were standardised to 1 mg of protein measured by the Bradford method [39]. The methodology and sample preparation [39,40,41,42] are described in detail in the Supplementary Material (S4).

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) and Tukey’s post hoc test were performed using STATISTICA 13.1 software (TIBCO Software Inc., Hillview Avenue, Palo Alto, CA, USA) at a significance level of α = 0.05. The statistical means of each experimental object (with standard deviation SD) were compared between experimental variants to analyse the results.

3. Results and Discussion

The content of bioactive compounds in E. sativa tissues showed significant changes when algal extract solutions were applied to the substrate in which the plants were cultivated.

3.1. Total Phenolic Compounds, Flavonoids, and Antioxidant Activity against DPPH

The phenolic content (Figure 1A) was significantly increased relative to the control at doses W2 and W3 when Ascophyllum nodosum-based solutions were applied. However, the phenolic content was significantly decreased when dose W4 (the most concentrated solution) was applied. The content of total phenolic compounds in young arugula significantly increased after the treatment with aqueous extracts of A. nodosum on the substrate. The most significant increase was observed at dose W2, which was 72% with respect to the control.

The flavonoid content in arugula microgreens almost doubled upon treatment with the extract at W2 and W3 doses. Furthermore, when the most concentrated dose of Ascophyllum nodosum (10%) was applied to the substrate, the flavonoid content in the experimental plant tissues was almost equivalent to those of control plants. The flavonoid content of young arugula increased significantly after application of aqueous algae extracts. The greatest increase was observed at dose W2 (89% relative to the control) (Figure 1B). It was demonstrated that the plants irrigated with the 2.5% solution had the highest antioxidant potential among the variants tested. Ascophyllum nodosum was also used in an experiment by Nikoogoftar-Sedghi et al. [43] which tested the effect of the foliar application of Ascophyllum nodosum on stress tolerance in pistachios. The results showed that the use of seaweed extract had a significant effect on total phenolic content, flavonoid content, and antioxidant activity. The content of TPC and flavonoids increased compared to the control, particularly at the highest concentration tested [43].

The experiment with aqueous seaweed extract of Ulva lactuca and Padina durvillei carried out by Osuna-Ruiz et al. [44] showed an increase in TPC in seedlings, particularly in treatments with low extract concentrations (0.5 mg mL⁻¹). TFC increased significantly in amaranth seedlings treated with 0.5 mg mL⁻¹ of P. durvillei compared to the control and all other concentrations. Flavonoid content increased significantly in sprouts treated with the highest concentration of U. lactuca. Interestingly, TPC showed a significant increase in treatments with low concentrations of seaweed extracts. Additionally, when a commercial product based on A. nodosum (ByoAlga^®) was used, TPC increased in parallel with the increase in product concentration. In the cited work, TFC increased after the application of Ulva lactuca and for low concentrations of Padina durvillei, but sprouts treated with commercial Ascophyllum showed significantly lower levels of flavonoids [44].

The antioxidant activity decreased with increasing extract dose (Figure 1C). The results for the highest extract concentration and the control were almost equal. The antioxidant activity against DPPH in young arugula increased significantly after the application of aqueous extracts of A. nodosum to the substrate. The greatest increase was observed at dose W2 (82% relative to the control). Ascophyllum nodosum extract significantly enhanced the antioxidant activity in young arugula plants.

The DPPH method is commonly used to evaluate the antioxidant activity in arugula due to its effectiveness in measuring the free radical scavenging capacity of phytochemicals. The study by Hamid et al. [45] evaluated the antioxidant potential of water, ethanolic and methanolic whole extracts of Eruca sativa seeds and oil, and their phytochemical composition. The antioxidant activity was evaluated by using the DPPH free radical scavenging protocol [42]. This method is particularly suitable for arugula as it allows for the quantification of the total free radical scavenging activity, which contributes to the variety of phytochemical constituents of arugula. The highest percentage of free radical inhibition (DPPH) was observed by Nikoogoftar-Sedghi et al. [43] after the use of seaweed extract (at each of the concentrations used) and the lowest in the control [43]. In the study by Arbos et al. [46], the antioxidant activity of the E. sativa methanol extracts (leaves) was determined using the DPPH assay. The total phenolic content in the extracts was also determined by the Folin–Ciocalteu method and calculated as mg equivalent of gallic acid. The antioxidant activity against the DPPH free radical was determined, i.e., in arugula and lettuce [46].

In a study on a similar species (Eruca vesicaria), Hassan et al. [47] used a commercial preparation also based on algae (TAM^®—a bio-fertiliser consisting of combined extracts from macroalgae species: Ulva lactuca, Jania rubens, and Pterocladiella capillacea). The researchers achieved the highest DPPH inhibition with a 10% extract which was slightly more effective than the control (NPK only). Conversely, the 5% and 15% solutions had a lower level of activity against DDPH compared to the control in 2016. However, in 2017, the 5% TAM^® formulation showed comparable results to the NPK treatment. Compared to the NPK control, 10% TAM^® showed the highest TAA (total antioxidant activity). The cited authors confirm that the concentration of the extract used can determine the antioxidant activity of arugula. According to the publication presented, the antioxidant activity measured against DPPH radicals (expressed in Trolox equivalents) was 4.7 mg TE g⁻¹ d.m. for the control and almost twice as much (8.59 mg TE g⁻¹ d.m.) for the W2 dose.

Agregán et al. [48] carried out a study to investigate the antioxidant capacity of extracts made from the brown macroalga, A. nodosum. The researchers used ultrasonic extraction as an innovative and eco-friendly method to prepare the extracts. The authors obtained algal extracts using water/ethanol (50:50, v:v) as the extraction solvent using ultrasound-assisted extraction. The extracts from Ascophyllum nodosum and Bifurcaria bifurcata exhibited the highest antioxidant potential compared to the other samples. In the present publication, UAE was also used, but only water was used as the solvent without the addition of ethanol.

Some marine algae contain several bioactive compounds that have been extensively studied for their potential effects on plant growth and stress tolerance. These compounds include phlorotannins, fucoxanthin, fucoidan, and other polysaccharides [49]. However, the specific mechanisms may require further research to be fully understood. It should also be taken into account that the process and methods used to prepare the extracts are crucial factors influencing the extract composition and therefore its properties.

The evaluation of the bioactive compound content in young rocket leaves is crucial because it directly translates into their biological activity. Lola-Luz et al. [50] conducted field experiments to evaluate the effect of a non-fortified Ascophyllum nodosum extract (ANE) on the yield and nutritional quality of two broccoli cultivars. Plants treated with seaweed extracts showed higher levels of total phenolic, flavonoid, and isothiocyanate contents compared to the untreated control group. The application of this algae extract also resulted in a significant increase in phenolic and flavonoid content in microgreens of Eruca sp. In the experiment conducted by Lola-Luz et al. [50], the broccoli cultivar ‘Ironman’ exhibited a 2.2-fold increase in total phenolic content and a 1.5-fold increase in total flavonoid content when exposed to a higher dose compared to the control group. The results indicate that higher doses have a positive effect on the phenolic and flavonoid content of these cultivars. Similarly, the cultivar ‘Red Admiral’ showed a 2.3-fold increase in total phenolic content and a 2.6-fold increase in total flavonoid content.

The inhibition of the biosynthesis of phenolic compounds and flavonoids in plants by higher concentrations of Ascophyllum nodosum algae may be attributed to the complex interaction of compounds present in the algae with plant metabolic pathways [51].

The study presented here confirms the findings of the cited authors [41] that seaweed extracts can significantly increase the amount of phytochemicals in plants, thereby improving their nutritional value. The phenolic content (expressed as gallic acid equivalent) after extract application increased from 2.66 mg GAE g⁻¹ d.m. in the control to 5.03 and 4.89 mg GAE g⁻¹ d.m. (for W2 and W3 doses, respectively). When the extract dose was increased to W4, the phenolic content decreased to a content similar to the control (2.43 mg GAE g⁻¹ d.m). This confirms the importance of determining the appropriate dose of extract (fertiliser, biostimulant) to modify the composition of microgreens. In a study by Elansary et al. [52], the phenolic and flavonoid levels, antioxidant capacity, and lipid peroxidation in plants were shown to increase significantly when treated with A. nodosum extract (soil and spray). This study evaluated Spiraea nipponica and Pittosporum eugenioides plants grown under controlled conditions. The results of that experiment showed an increase in phenolic and flavonoid content in response to the application of A. nodosum extracts, which also seems to corroborate the authors’ findings. In a publication by Bajpai et al. [53], the treatment of strawberry plants with Ascophyllum nodosum extract was shown to alter the phenolic and flavonoid content of powdery mildew-infected strawberry leaves. The cited study results indicate that plants treated with ANE had a significantly higher flavonoid content than the control plants. Leaves sprayed with 0.2% extract had the highest flavonoid content after 120 h, which was 47.2% higher than the control group. In addition, leaves sprayed with 0.2% A. nodosum extract showed a maximum increase in total flavonoid content 120 h after inoculation with Podosphaera aphanis. Spraying with the extract induced phenolic production in strawberry plants. Total phenolic content increased by 30%, 51.5%, and 14%, respectively, in strawberry plants sprayed with 0.1%, 0.2%, and 0.3% A. nodosum extract compared to the control 120 h after inoculation with P. aphanis spores [53].

Plants have undoubtedly evolved an impressive array of phenolic secondary metabolites. Although not essential for primary growth and development, these metabolites are crucial for the plant’s interaction with the environment, reproductive strategy, and defence mechanisms [54]. Phenolic compounds, in particular, are an important class of plant secondary metabolites that play critical physiological roles throughout the plant life cycle. Plants synthesise more phenolic compounds, including phenolic acids and flavonoids, when faced with abiotic stress. These compounds help plants to adapt more efficiently to environmental conditions. Under abiotic stress conditions, the phenylpropanoid biosynthetic pathway is activated, leading to the accumulation of various phenolic compounds that effectively scavenge harmful reactive oxygen species [55]. These compounds have multiple functions highlighting the importance of their role in mitigating the effects of abiotic stress. Phenolic metabolism in plants plays an important role in providing aromatic amino acids, defence-related compounds, chemical attractants or repellents, structural support, UV protection, and colour. The combined action of the shikimate and phenylpropanoid pathways is responsible for the biosynthesis of phenolics in plants [54]. Under stress conditions, the biosynthesis of phenolic compounds is regulated by changes in the activity of key enzymes in the phenol biosynthetic pathways. These enzymes include PAL which catalyses the first step in the biosynthesis of phenolic compounds in plants and acts as a key regulatory enzyme in specialised metabolism, and PPO which catalyses the oxidation of monophenols and/or o-diphenols to o-quinones [56,57,58]. PAL is the first enzyme in the general phenylpropanoid pathway, catalysing the non-oxidative elimination of ammonia from L-phenylalanine to give trans-cinnamate [59]. Plant polyphenol oxidases (PPOs) are widespread and well-studied oxidative enzymes. Their effects on discolouration in damaged and diseased plant tissues have been known for many years [60]. In summary, PAL and PPO are enzymes involved in the phenolic pathway which plays a key role in plant defence and stress response. These enzymes affect the synthesis of phenolic compounds including flavonoids in plants, particularly under abiotic stress conditions [55].

3.2. Phenylalanine Ammonia Lyase and Polyphenol Oxidase Activities

The activity of selected enzymes responsible for the biosynthesis and conversion of bioactive compounds was also measured in the present experiment: PPO (Figure 2A) and PAL (Figure 2B).

PPO activity increased significantly when the substrate in which the plants were grown was treated with an extract of A. nodosum. The greatest increase was observed at dose W3 (84.2% relative to the control). It was demonstrated that there was an increase in the activity of these enzymes in arugula microgreens of the specimens that were treated with Ascophyllum nodosum extracts. The highest activity of PAL and PPO was observed after the treatment of Eruca sativa plants with the W3 dose. PAL activity increased to an extreme extent after the application of aqueous extracts of algae to the substrate. The greatest increase was observed at dose W3 (1213.5% relative to the control). In the case of both enzymes tested, an application of the highest dose (W4) did not increase their activity. In any case, under the treatment with Ascophyllum nodosum extracts, the activity of the enzymes tested responsible for the biosynthesis of bioactive compounds (PAL) increased in young leaves or microgreens of E. sativa. This is consistent with data reported in the literature based on experiments with other plants. Bajpai et al. [53], studying strawberry plants, showed that A. nodosum extracts upregulated enzymes associated with defence against powdery mildew infection. This study investigated the effect of using seaweed extracts on the induction of the defence enzymes PAL, PO, and PPO in strawberry leaves. Non-inoculated plants treated with ANE by foliar spraying showed increased PAL activity. The highest PAL activity was found in plants treated with 0.2% extract. The inoculation of the pathogen on sprayed leaves also increased PAL activity. Maximum PAL induction was observed after 120 h from inoculation in plants treated with 0.2% A. nodosum extract. The treatment of plants with A. nodosum extracts was found to induce defence-related enzymes and increase plant resistance to powdery mildew infection. A study by Ali et al. [61] investigated how the application of A. nodosum extract affected diseases in field tomatoes. Plants treated with the seaweed extract showed significantly increased levels of defence enzyme activity (PPO, PAL, PO, chitinase, glucanase) and accumulated higher amounts of phenolics than the control group. Examination of the transcript levels of marker genes for the defence pathway showed a higher expression of the JA/ethylene pathway compared to the SA pathway. Jayaraman et al. [62] conducted a study to evaluate the effect of a commercial A. nodosum-based product (Stimplex™) on the amount of defence enzymes and the total phenolic content in cucumbers. Chitinase, glucanase, PO, PPO, PAL, and lipoxygenase levels were determined in extracts from A. nodosum-treated cucumber plants. This study showed that the enzyme activity was higher in plants treated with Stimplex™ than in the control group. Plants that were sprayed or soaked with the Ascophyllum nodosum-based formulation showed increased levels of phenolics compared to the control group [60]. Transcripts of defence genes such as chitinase, lipoxygenase, glucanase, PO, and PAL genes were assessed. Plants treated with A. nodosum accumulated defence gene transcripts at a higher level than the control group [63]. Induced PO activity was also demonstrated in pepper plants sprayed with Ascophyllum nodosum extract. A single application of the extract at 0.8 or 1.6 L·ha⁻¹ stimulated PO activity while a double application resulted in an eightfold increase in PO activity [51,52]. Abkhoo and Sabbagh [64] found that cucumber plants sprayed with a commercial Ascophyllum nodosum extract showed increased activity of several defence-related enzymes such as β-1,3-glucanase, peroxidase, and polyphenol oxidase. They also noted changes in the transcript levels of several defence genes such as pathogen-induced lipoxygenase, phenylalanine ammonia lyase, and galactinol synthase in plants treated with the A. nodosum extract. Cucumber plants treated with this extract accumulated more phenolics than the control plants. This suggests that brown algae extracts may enhance disease resistance in cucumber by inducing defence enzymes and genes. A study by Rinaldi et al. [65] measured the efficacy of an A. nodosum extract (75 g L⁻¹) in reducing nematode populations in pathogen-infected soybean plants. In addition, the activation of defence enzymes in inoculated and non-inoculated plants was assessed. The enzymes peroxidase (PO), PPO, phenylalanine ammonia lyase (PAL), and glucanase showed the highest activity in plants treated by immersion and not inoculated 12 days after treatment. The results have also showed that the investigated extract can also potentially increase the activity of enzymes involved in the plant defence system compared to both the untreated and inoculated control groups. In an experiment described by Patel et al. [66], the combined application of A. nodosum extract and chitosan to pea plants increased the activity of the plant’s defence enzymes (PAL, PO) and the production of ROS. The treatment also resulted in an increased expression of many plant defence genes in the jasmonic acid (JA) pathway. The treatment of pea plants with a combination of A. nodosum extract and chitosan increased the expression of genes involved in the defence processes such as NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and innamate 4-hydroxylase. In addition, Stimplex™ treatment (based on A. nodosum) improved the activities of chitinase, glucanase, PO, PPO, PAL, and lipoxygenase enzymes in cucumber leaves [62]. In another experiment, carrot plants grown under greenhouse conditions were sprayed with a 0.2% A. nodosum extract and then exposed to fungal diseases (Alternaria radicina and Botrytis cinerea). Plant-defence-related enzymes were measured, including PO, PAL, chitinase, and β-1,3-glucanase. The activity of these enzymes was significantly increased in plants treated with the macroalgae extract compared to the control. These results suggest that using A. nodosum extract may help to strengthen the carrot plants’ defence system against disease by activating defence proteins or genes [67]. In the present study, PAL activity in the microgreens of arugula was observed to increase several-fold under the application of the extracts. For the control, this activity (measured by detection of t-cinnamic acid after enzymatic reaction with phenylalanine) was 0.37 U mg⁻¹, whereas after the application of extract concentrations at the doses W2 and W3, it was 3.48 and 4.86 U mg⁻¹, respectively. Once this value was reached, the PAL activity did not increase with increasing extract concentrations. Simultaneously, the value of dose (concentration) W4 was 1.6 U mg⁻¹. Changes in bioactive compound levels in plant material are attributed to the activation of enzymatic systems responsible for biosynthesis in plants [68]. Fertilisers and other abiotic and biotic factors can stimulate this activity during plant growth and development. The increase in total phenolic content in young leaves or microgreens found in studies is likely to be due to the activation of the PAL, which takes part in the biosynthesis of phenolic compound precursors. The most pronounced changes in the activity of PAL were observed in the leaves of young arugula irrigated with W3 extract. Other researchers have also identified similar variations in PAL and PPO activity as well as the phenolic compound content of the raw material produced. Migut et al. [69] found that the application of antioxidants in various forms affected the activity of different enzyme systems, including PPO, resulting in the reduced degradation of phenolic compounds in maize plants.

The authors speculate that the application of W2 dosage contributes to balance the metabolism of phenolic compounds (biosynthesis vs. degradation). The highest levels of phenolic compounds were observed in plants irrigated with the W2 extract. Although increasing the dose of the extract stimulates PAL biosynthesis, no further increase in total phenolics is observed, which may be due to the depletion of substrates involved in their biosynthesis, e.g., phenylalanine. On the other hand, the correlative increase in PPO induced by the abiotic stress caused by PAL may intensify the degradation of phenolics (especially those with a simple structure), resulting in a decrease in the level of TPC.

3.3. Catalase and Superoxide Dismutase Activities

The enzymatic mechanisms for ROS scavenging in plants comprise various enzymes including SOD, ascorbate peroxidase (APX), glutathione peroxidase (GPX), and CAT, as cited in Miller et al., 2019 [70]. However, the most commonly used markers of plant resistance are SOD and CAT. The experiment also assessed the activity of enzymes responsible for ROS uptake: CAT (Figure 3A) and SOD (Figure 3B).

It was observed that the application of A. nodosum extract increased the levels of these enzymes. CAT activity in arugula microgreens increased significantly after the application of aqueous extracts of algae to the substrate. The greatest increase was observed at dose W3 (68.2% relative to the control). SOD activity increased by 25% after the application at dose W4 compared to the control. The results of other studies also suggest that Ascophyllum nodosum extract increases the levels of the enzymes SOD and CAT in plants [71]. SOD and CAT are ROS-scavenging enzymes that help protect plants from oxidative stress. The presence of abiotic stressors can induce an increased production of low-molecular-weight antioxidants, including flavonoids and phenolic compounds, in which PAL (Figure 2A) is involved in their biosynthesis. The antioxidant potential measured by the DPPH method is also influenced by these compounds (Figure 1C). The results obtained are in agreement with those obtained by Nikoogoftar-Sedghi et al. [43] for pistachios (significantly higher SOD and CAT activity compared to the application of A. nodosum extract). In the cited work, the activity of SOD and CAT was much higher compared to the control but the different concentrations of the extracts (and the method of obtaining them) should be taken into account.

When lettuce plants [72] were cultivated hydroponically, the effects of foliar applications of either extracts of A. nodosum seaweed or potassium salts were evaluated. The results indicate that a decrease in plant biomass, photosynthetic rate, potassium content, and tissue antioxidant capacity, as well as leaf stomatal conductance, occurred in lettuce plants with low potassium salt levels compared to those with higher potassium levels. The application of A. nodosum extract helped to overcome potassium deficiency and increased plant tissue antioxidant capacity, including activities of SOD and CAT [72]. The use of Ascophyllum nodosum extract is known to enhance drought tolerance by reducing ROS-induced malondialdehyde production in beans (Phaseolus vulgaris) through improved CAT activity [71]. Foliar sprays of A. nodosum extract similarly reduced lipid peroxidation in Paspalum vaginatum grown under long-term irrigation conditions [73]. Reduced levels of ROS in P. vaginatum grown under drought stress conditions were linked to several factors including a greater activity of antioxidant enzymes like SOD and CAT. The production of ascorbate, a non-enzymatic antioxidant, was increased [19,64]. The application of a commercial seaweed extract (Tasco^®, Acadian Seaplants Limited, Dartmouth, Canada) significantly increased the activity of the antioxidant enzyme SOD in unstressed turf grasses [74]. Thus, the application of commercial seaweed extract to tall fescue in these experiments seems to primarily increase its antioxidant capacity [75].

Kandasamy et al. [76] investigated heat stress tolerance in animals using Tasco^®. The aqueous extract of the formulation was shown to improve heat stress tolerance and prolong the life span of Caenorhabditis elegans. Increased levels of SOD and glutathione peroxidase were found. There has also been research [77] analysing the ability of A. nodosum and humic acid extracts to improve antioxidant status, drought and disease tolerance, and growth response when applied to leaves of tall fescue (Festuca arundinacea Schreb) and Kentucky bluegrass (Poa pratensis L.). The application of Ascophyllum nodosum extract increased SOD activity, photosynthetic capacity, and chlorophyll content in tall fescue and Kentucky bluegrass (Agrostis palustis Huds.). Infection with endophytes had no significant effect on SOD activity, photosynthetic capacity, or chlorophyll content. A correlation between SOD and photosynthetic capacity was found in summer in both endophyte-free and endophyte-infected tall fescue [77].

The exact molecular basis for the improved growth and adaptation to stress induced by the use of A. nodosum extracts is difficult to elucidate. This is partly due to the polygenic response involved in these complex and dynamic processes, and the difficulty to distinguish between direct and secondary effects [78].

The results show that application of aqueous extracts of Ascophyllum nodosum to the substrate increases the total phenolic content, flavonoid content, and antioxidant activity in young arugula leaves. It also favours the activity of plant defence enzymes. This may be due to the presence of various bioactive compounds in the extracts which may have a positive effect on plant growth and stress tolerance. A. nodosum extracts increased the antioxidant activity of arugula microgreens, indicating that the extracts may contain compounds that help scavenge ROS and protect plants from oxidative stress.

Algae-extract-based biostimulants have been shown to prime plants and crops. This process involves molecular priming that modulates genes and pathways in plants through the timely application of specific factors. As a result, plants gain durable tolerance to abiotic and oxidative stress. Products based on seaweed extracts have been shown to induce desired changes in plant signalling cascades and metabolic processes, resulting in improved plant growth and crop performance. The precise pathways activated by seaweed extracts and their individual bioactive compounds and/or their synergism may be responsible for the above-described effects in plants, but further studies at the molecular level are required [79].

Macroalgae extracts have been found to possess biostimulant properties that can enhance plant growth, productivity, and stress tolerance. One of the key species used as a source of biostimulants is Ascophyllum nodosum. The mechanism of action of A. nodosum extracts on plants is currently thought to be multi-pathway. A. nodosum extracts contain minimal concentrations of minerals that plants can readily assimilate, but their primary benefit is their ability to stimulate various processes in the plant’s metabolic system. A. nodosum extracts can elicit various responses in plants to combat biotic and abiotic stresses. They also affect plant metabolism and signalling pathways that are specifically linked to nutrient uptake or translocation. Research has demonstrated that such factors can decrease abiotic and oxidative stress in plants, resulting in enhanced nutrient utilisation efficiency and productivity and improving quality traits. In summary, A. nodosum extracts stimulate various processes in plants, eliciting multiple responses, influencing plant metabolism and signalling pathways, and leading to greater resistance to biotic and abiotic stresses. However, the genetic, physiological, and biochemical mechanisms are not fully understood and require comprehensive and systematic research.

4. Conclusions

This study examined the impact of raw, aqueous extracts of a valuable alga Ascophyllum nodosum on the intensification of biosynthesis antioxidant compounds such as phenolic compounds (incl. flavonoids), using arugula as an example. The extracts were environmentally friendly and additive-free.

The experiment demonstrates the following:

• The total phenolic content of arugula microgreens was found to increase significantly following the application of aqueous extracts of A. nodosum to the substrate. The greatest increase (72%) was observed at a concentration of 2.5%.
• The flavonoid content was found to increase significantly following the application of aqueous extracts of seaweed. The greatest increase was observed at a concentration of 2.5% with a value of 89%.
• The antioxidant activity (DPPH) demonstrated a notable increase following the application of aqueous extracts of A. nodosum. The greatest increase was observed at a concentration of 2.5% (82%).
• The application of aqueous extracts of algae to Eruca sativa microgreens resulted in a notable elevation in CAT activity. The greatest increase was observed at a concentration of 5% with a value of 68.2%.
• The application of A. nodosum extract resulted in an increase in SOD activity. The greatest increase was observed in dose W3 which equates to a concentration of 5% (25% increase).
• PAL activity increased to a considerable extent following the application of aqueous extracts of Ascophyllum nodosum. The greatest increase was observed at dose W3 (1213.5% relative to the control).
• PPO activity increased significantly when the substrate in which the plants were grown was treated with algal extracts. The greatest increase (84.2%) was observed at a concentration of 5%.

In conclusion, this research supports the use of algae extracts as a sustainable and effective approach to improve the growth, quality, and stress tolerance of arugula plants, making them a valuable fertiliser and elicitor in the cultivation of arugula microgreens. The use of organic and environmentally friendly extracts in the cultivation of microgreens is particularly important as it ensures that the produce is free from harmful chemicals and pesticides, making it healthier to eat. Microgreens, in particular, can be grown without fertilisers or pesticides due to their short growing season which can contribute to a more sustainable and healthier food system.