Influence of Vermicompost Tea on Metabolic Profile of Diplotaxis muralis: An NMR Spectroscopic Analysis

Abstract

Recently, we reported the efficacy of organic nutrient solutions in supporting the hydroponic cultivation of Diplotaxis muralis. The aim of this study was to elucidate the influence of standard and enhanced vermitea formulations, compared to the conventional Hoagland solution, on phytochemical and metabolomic changes in D. muralis. Using NMR-based metabolomics and multivariate analysis, we observed significant metabolite variation among treatments. Both vermitea formulations increased the levels of acetate, alanine, and 2-oxoglutarate, and boosted the biosynthesis of key secondary metabolites, including methoxy flavonoids and glucosinolates. The standard vermitea treatment further resulted in a higher accumulation of leucine and citrate, while the Hoagland solution induced higher glucose concentrations. Enhanced vermitea improved copper and zinc uptake, positively correlating with methoxy flavonoid production. In contrast, the higher phosphorus and potassium content of the Hoagland solution correlated with increased glucose levels in D. muralis. Metabolite profiling coupled with multivariate analysis identified the enhanced vermitea as the best alternative to chemical nutrient solution for improving the nutritional and phytochemical quality of D. muralis leaves.

1. Introduction

Salad crops from the Brassicaceae family, collectively known as rocket, rucola, or arugula, are native to Mediterranean countries, specifically those in the western region. This area is considered a center of diversity due to its unique climatic conditions [1]. Rocket crops comprise various species from the Eruca and Diplotaxis genera. The Diplotaxis genus includes a group of herbaceous annuals, typically diploid, with rare instances of polyploidy and dysploidy. D. muralis, also known as the annual wall rocket, is an allotetraploid produced by the hybridization of D. viminea and D. tenuifolia. The D. muralis is used in Mediterranean cuisine as a salad ingredient and garnish due to its distinctive pungent and peppery flavor [2]. The organoleptic properties of D. muralis, such as its spicy flavor, are attributed to a rich array of bioactive compounds, including glucosinolates (GSLs) and flavonoids. The hydrolysis of GSLs by myrosinase results in the production of isothiocyanates, thiocyanates, and nitriles [3]. These compounds are believed to possess numerous health-promoting potentials, such as antioxidant and anticarcinogenic properties [4]. Rockets are effective in the prevention of various diseases, including coronary heart disease and inflammatory disorders [5].

Regarding the agricultural production of Diplotaxis species, these are commonly grown in open fields but can also be cultivated in soilless systems. Soilless culture systems produce clean, fresh vegetables in a significantly shorter time compared to traditional cultivation and facilitate postharvest processes in the industry. However, the environmental impact resulting from the disposal of hydroponic solutions after the crop growth cycle cannot be overlooked [6]. In hydroponics, chemical fertilizer salts are dissolved in water to create nutrient solutions essential for plant growth. These nutrient solutions need to be replaced periodically, resulting in the production of hydroponic wastewater, which is often disposed of directly into the environment, causing contamination [7]. Therefore, the use of organic nutrient solutions as a potential alternative to inorganic nutrient solutions in hydroponic farming is crucial [6]. The ability to transform diverse organic waste into organic fertilizers enriched with all essential elements for plant growth has made vermicompost a viable option for sustainable agriculture [8]. The aqueous extract of vermicompost, known as vermicompost tea or vermitea, contains nutrients, microbes, and phytohormones beneficial to plant growth when applied as a foliar application or in the soil [9]. The symbiotic relationship between plants and microbes enhances the production of phytohormones and nutrient acquisition [10]. Microbes have the potential to chemically communicate with their host plants, stimulating the immune system and affecting the plant’s physiology and health [11], thereby influencing the plant’s metabolism. Moreover, the application of vermicompost influences plant metabolites such as phenols, flavonoids, and various amino acids [12,13].

In our previous study, D. muralis plants were cultivated using three different hydroponic solutions: Hoagland solution, standard vermitea, and enhanced vermitea [14]. The variations in pH, electrical conductivity (EC), and the nature of the nutrient solutions (organic vs. chemical) significantly influenced the plant growth parameters and the uptake of macro and micronutrients in the leaves of D. muralis. These variations in plant growth parameters and nutrient acquisition can affect the metabolic profile of the plant. Primary metabolites serve nutritional purposes, whereas secondary metabolites are used by plants as a defense against various biotic and abiotic stresses [15]. The effect of different agronomic practices, such as water supply and nitrogen nutrition, on quality traits and metabolism of different rocket species has been confirmed by several studies [16,17,18]. A recent study by Buitrago-Villanueva et al. [19] assessed the impact of the culture system and harvest time on metabolite composition in Eruca sativa leaves, reporting that some compounds were significantly altered by the culture system. These studies explored the metabolite composition, mainly focusing on GC-MS and LC-MS metabolomic studies. NMR-based metabolomics is a powerful approach to studying plant metabolism, providing comprehensive insights into the chemical composition of plant systems. Several studies have employed an NMR-based metabolomic approach to evaluate changes in the metabolic profiles of edible plant parts [20,21,22]. NMR spectroscopy provides comprehensive information on metabolite composition in complex plant samples and is widely applied in food science for traceability, authenticity, and safety studies [23].

In this context, to investigate sustainable farming approaches for D. muralis production and its significance as an important food source, three different nutrient solutions were used in hydroponic cultivation: Hoagland solution (HS), standard vermitea (SVT), and enhanced vermitea (EVT). The impact of different growth conditions on the metabolite pattern of rockets, specifically D. muralis, using nuclear magnetic resonance (NMR) spectroscopy, has not been previously characterized. The present investigation employs ¹H NMR spectroscopy to determine the metabolic composition of D. muralis to understand the bioactive chemicals associated with the type of plant growth medium. These findings will enhance our understanding of the biochemical composition of Diplotaxis species and contribute valuable data to the broader field of plant metabolomics. The insights gained could pave the way for new therapeutic applications and enhance the nutritional profile of D. muralis.

2. Materials and Methods

2.1. Experimental Design

The preparation of SVT and EVT and their physicochemical properties are reported in our previous study [14]. SVT was prepared by mixing green waste vermicompost (VC) with double-distilled water (DDW) in a 1:10 ratio. The pH of SVT ranged between 7.5 and 8.0, and no further pH alteration was made. The electrical conductivity (EC) of SVT was 1.292 mS/cm. EVT, on the other hand, was prepared by mixing VC with DDW in a 1:5 ratio. The pH of EVT was maintained within the range of 5.80–6.30 by gradual addition of a few drops of diluted sulfuric acid (10% H₂SO₄). The EC of EVT was approximately 2.0 mS/cm. The higher EC value can be interpreted as indicating higher nutrient content in the growth media, due to the presence of more dissolved nutrients, specifically ions like NO₃⁻, K⁺, and PO₄³⁻, in solution with a high EC value. Therefore, SVT and EVT are differentiated by pH value and nutrient concentration. Both organic nutrient solutions (SVT and EVT) can contain various phytohormones and beneficial microbes (which are absent in chemical nutrient solutions) that influence plant growth. The pH and EC of EVT and Hoagland solution (HS) are comparable; however, the nutrient composition varies between the two solutions due to the differences in their organic and chemical sources, respectively.

D. muralis seeds were germinated in plastic Petri dishes containing a double layer of moist filter paper. Following six days of germination, five sprouted seeds were carefully transferred into each plastic pot filled with perlite. These pots were then placed into a hydroponic system. Fresh hydroponic solutions, such as SVT, EVT, and Hoagland solution, were circulated through the pots to ensure a consistent supply. An aquarium pump was used to maintain an adequate supply of oxygen. The pH of EVT and Hoagland solution was adjusted to a range of 5.80–6.30 using diluted sulfuric acid and was continuously monitored every two days. To prevent nutrient depletion, the hydroponic solutions were replaced with fresh nutrient solutions every two weeks. A completely randomized design (CRD) experiment consisting of three treatments and six replications was employed. The plants were grown in a growth chamber at the University of Salento, Lecce, Italy (40°20′12″ N; 18°07′24″ E) under controlled conditions (25/18 °C day/night temperature, 14 h of daylight, and 65% humidity). Leaves of D. muralis were harvested after 45 days, at the onset of flowering.

2.2. Sample Preparation for NMR Metabolomics Analysis

Samples were prepared according to a previously reported experimental method [24]. Specifically, D. muralis leaf samples were rapidly frozen in liquid nitrogen, pulverized to a fine powder using a stainless-steel blender, and subsequently lyophilized for 48 h. For metabolite extraction, 0.75 mL of CD₃OD and 0.75 mL of KH₂PO₄ buffer in D₂O (pH 5.9) containing 0.05% w/v TSP-d4 (sodium salt of trimethylsilylpropionic acid) were combined with plant material (100 mg). The mixtures were carefully vortexed at room temperature for 1 min, followed by 10 min of sonication under the same conditions. Samples were centrifuged at 17,000× g for 20 min, and 700 µL of the resulting supernatant was transferred into a NMR tube (5 mm).

2.3. NMR Measurements and Metabolite Identification

All measurements were conducted using a Bruker AVANCE III 600 Ascend NMR spectrometer (Bruker, Ettlingen, Germany), operating at 600.13 MHz for proton detection. The spectrometer was fitted with a TCI cryoprobe that included a z-axis gradient coil and automatic tuning/matching (ATM). The experiments were conducted at 300 K under automation, with samples introduced via a Bruker Automatic Sample Changer integrated with IconNMR software “Version 5” (Bruker) for sample loading. Each spectrum was acquired using a 1D sequence with pre-saturation, and a composite pulse for selection (zgcppr, a standard pulse sequence from Bruker) was executed, comprising 16 transients and 16 dummy scans, with a relaxation delay of 5 s. The free induction decay (FID) data consisted of 64 K data points, with a spectral width of 12,019.230 Hz (20.0276 ppm) and an acquisition time of 2.73 s. Prior to Fourier transformation, the resulting FIDs were processed with an exponential weighting function corresponding to a line broadening of 0.3 Hz, followed by automated phasing and baseline correction [25].

Metabolites were identified through ¹H and ¹³C assignments by 1D and 2D homo- and heteronuclear experiments (Figures S1–S3), supported by comparison with other reported literature [26,27,28,29]. ¹H NMR data processing was performed by using TopSpin 3.6.1 (Bruker) and Amix 3.9.15 (Bruker, BioSpin, Italy) for visual inspection and the bucketing process for further multivariate statistical analyses. All spectra were referenced to the TSP signal (δ = 0.00 ppm).

2.4. ¹H NMR Data Processing and Multivariate Statistical Analysis

The ¹H NMR chemical shifts corresponding to the identified metabolites are presented in Table S1. The bucketing preprocessing procedure was utilized for the ZGCPPR spectra across the 10.0 to 0.5 ppm region. Each NMR spectrum was systematically divided into rectangular buckets of fixed width, measuring 0.04 ppm, and integrated using Bruker Amix 3.9.15 software (Bruker, BioSpin).

The remaining buckets were normalized to the total area to reduce minor discrepancies and then mean-centered. Multivariate statistical analyses, including unsupervised principal component analysis (PCA) and supervised orthogonal partial least squares discriminant analysis (OPLS-DA), were conducted to investigate the inherent variation within the data, utilizing SIMCA 14 software (Sartorius Stedim Biotech, Umeå, Sweden) [30,31]. The Pareto scaling method was applied by dividing the mean-centered data by the square root of the standard deviation. The robustness of the statistical models was tested by the cross-validation default method (7-fold) and further evaluated with a permutation test (100 permutations). The total variations in the data and the internal cross-validation, hence the quality of the statistical models, were described with R²(cum) and Q²(cum) parameters and p values (p[CV-ANOVA]) (a p-value of < 0.05, confidence level of 95%, was considered statistically significant) obtained from analysis of variance testing of cross-validated predictive residuals (CV-ANOVA).

2.5. Chemicals

Deuterium oxide (99.9 atom%D) containing 0.05% wt 3-(trimethylsilyl)propionic-2,2,3,3 d4 acid sodium salt (TSP), and potassium phosphate monobasic was purchased from Armar Chemicals (Döttingen, Switzerland). Methanol-d4 (99.9 atom %D) and potassium phosphate monobasic were purchased from CARLO ERBA Reagents (Milan, Italy).

3. Results

3.1. ¹H-NMR Characterization of D. muralis Metabolic Extract

The metabolic variation induced by the different nutrient solutions was analyzed using NMR-based metabolomic approaches. The ¹H NMR spectra of the leaves’ hydroalcoholic extracts, for each treatment condition (HS, SVT, EVT), were collected, and a multivariate statistical analysis was subsequently applied. The representative ¹H NMR spectrum, Figure 1, and the primary resonances were assigned to individual metabolites and are reported in Table S1.

A total of 28 compounds were identified through 1D and 2D NMR spectral analysis and compared with previously reported data [24]. The spectrum is characterized by a complex pattern of signals attributable to sugars (α and β glucose), amino acids (alanine, glutamate, glutamine, histidine, isoleucine, leucine, threonine, asparagine, arginine, phenylalanine, tyrosine, glycine, γ-amino-butyrate, and valine), organic acids (citric acid, formic acid, fumaric acid, malic acid, and succinic acid), flavonoids (Kaempferol analogues, quercetin analogues), glucosinolates (glucoraphanin, neoglucobrassicin) and others (adenine, choline). Notably, the spectra of D. muralis exhibit pronounced peaks corresponding to flavonoids and glucosinolates. The antioxidant activity of Brassicaceae leaves is closely correlated with their flavonoid content, whereas glucosinolates play multifaceted roles, including key functions in plant defense [32,33].

3.2. Multivariate Statistical Analysis

A preliminary unsupervised analysis (PCA) was performed on the entire dataset to observe a general trend of data clustering (Figure 2). Bucket tables obtained from the aqueous leaves’ extracts (¹H ZGCPPR NMR spectra) were used as input variables. A two-component PCA model describes 68.9% of the total variance of the dataset. Samples grouping along the t[1] component of the PCA score plot model was observed (Figure 2), particularly evident among HS and SVT samples, indicating a significant difference in the metabolic profile of D. muralis depending on the different growth conditions.

Further supervised OPLS-DA analysis was applied to the NMR bucketed data to discriminate between the predefined groups of samples by separating predictive (correlated with class separation) from orthogonal components (unrelated to the class separation). This approach aimed to improve the interpretability of the model and simplify the identification of key variables contributing to class differences. Pairwise supervised OPLS-DA analysis performed on leaf samples’ extracts according to the variable “growth conditions”, i.e., Hoagland solution vs. standard vermitea and Hoagland solution vs. enhanced vermitea, yielded good models (1 + 1 + 0) with significant predictive capability Q² = 0.87 and Q² = 0.69, respectively (Figure 3B,D), thus reflecting more differentiation among the variables.

The score scatter plots of the two OPLS-DA models (Figure 3A,C) demonstrated a clear separation in each pairwise comparison (HS vs. SVT and HS vs. EVT). The corresponding S-line plots identified the metabolites responsible for the observed class separation between HS vs. SVT and HS vs. EVT (Figure 3B,D). From our analysis, the SVT and EVT growth conditions induce higher levels of acetate, alanine, 2-oxoglutarate, flavonoids (especially methoxy flavonoids comparable to quercetin analogues in which the hydroxy group is replaced by a methoxy group), and glucosinolates (glucoraphanin, neoglucobrassicin), and lower levels of glucose compared to HS. Furthermore, SVT also enhances the content of leucine and citrate in comparison to HS.

The Box-and-Whisker plots, Figure 4, illustrate the alteration trends of significant metabolites, identified in the OPLS-DA pairwise comparison models, concerning the different treatment conditions (SVT, EVT, HS). Further, one-way ANOVA with Tukey’s post hoc test revealed leucine, glucose, 2-oxoglutarate, methoxy flavonoids, citrate, acetate, and alanine as highly significant metabolites (p < 0.05). Glucosinolates did not show highly significant changes (p > 0.05).

To correlate the content of discriminant metabolites in D. muralis grown under different hydroponic conditions and the accumulation of metals, as previously studied [14], an additional partial least squares (PLS) regression model was built (4 components, R2X(cum) = 0.788, R2Y (cum) = 0.926, Q2 (cum) = 0.595), as shown in Figure 5. The predictor variables (loadings) were related to the metal content (Cu, Zn, Fe, Mn, P, Ca, and K), quantified by ICP-AES [14]. The loading plot, Figure 5B, and its expansion, Figure 5C, display how the predictor variables contribute to the PLS components. The axes (w*c [1] and w*c [2]) represent how strongly each variable is correlated with the first two PLS components, highlighting that the accumulation of Fe, Mn, P, Ca, and K is associated with a higher amount of glucose in HS samples compared to the SVT, which was characterized by a higher content of methoxy flavonoids (quercetin analogues) and lower content of Fe, Mn, P, Ca, K, Cu, and Zn. EVT samples showed an intermediate profile between SVT and HS. The elevated concentration of methoxy flavonoids observed in EVT samples is associated with increased levels of Cu and Zn. Additionally, higher concentrations of Fe and Mn were also detected in EVT. The HS samples are characterized by lower content of Cu and Zn, which, from our analysis, is related to the higher content of glucose and K compared to SVT.

4. Discussion

In the present study, the variations in the metabolic profile of D. muralis, influenced by different growth media, HS, SVT, and EVT, were evaluated. The characterization of D. muralis leaves grown in organic (vermiteas) and chemical nutrient solutions using nuclear magnetic resonance (NMR) spectroscopy, along with multivariate data analysis, was provided. A preliminary unsupervised principal component analysis (PCA) performed on the entire dataset described the variability due to different growth conditions, resulting in a clear differentiation in D. muralis primary and secondary metabolites. The type and concentration of plant metabolites are greatly affected by crop type, cultivar, culture environment, and fertilization level [34]. The PCA described a higher degree of separation between HS and SVT as compared to HS and EVT. This effect may be due to the significant differences in pH and nutrient contents between the HS and SVT hydroponic solutions. The differences in the pH of growth media can alter the accumulation of secondary metabolites [35]. Besides, SVT contains low levels of essential plant nutrients such as nitrogen, phosphorus, potassium, and other micronutrients. Such deficiencies may disrupt the regulation of metabolic pathways, leading to alterations in the metabolite profile [19]. In contrast, EVT contains an adequate concentration of nutrients to meet the nutritional demands of rocket plants. Additionally, the pH values of both EVT and HS were maintained at similar levels as required for optimal plant uptake [14].

To further explore the variation in metabolites, pairwise OPLS-DA comparisons (HS vs. SVT and HS vs. EVT) with their corresponding S-line models were performed (Figure 3). As described by significant predictive capability (Q2) values, clear discrimination was observed in HS vs. SVT and HS vs. EVT comparisons, suggesting the influence of vermitea on the metabolite patterns in D. muralis. Other authors have also confirmed the impact of vermicompost on the metabolic profiles of Moringa oleifera [36] and Ocimum basilicuum leaves [37]. However, the alterations in the metabolite profile of soilless-grown D. muralis in response to vermicompost tea (vermitea) have not been studied previously.

Vermicompost possesses plant biostimulatory activity due to the presence of phytohormones, organic acids, fulvic acids, humic acids, and beneficial microbes [14,38]. These plant biostimulants may influence the production of secondary metabolites [39]. Türkay and Öztürk [40] investigated the effects of solid vermicompost and vermitea on phenylpropene biosynthesis in basil (Ocimum basilicum L.) plants. Their findings revealed that the application of vermitea significantly enhanced the levels of phenylpropenes, which are key compounds that contribute to the characteristic aroma and antimicrobial activities of basil. The positive influence of vermicompost on the flavonoid content and antioxidant capacity and antioxidant capacity of various plant species has also been reported [41,42].

The overall composition of plant metabolites reflects the different metabolic pathways. Metabolites such as acetate, alanine, 2-oxoglutarate, flavonoids (especially methoxy flavonoids comparable to quercetin analogues in which the hydroxy group is replaced by a methoxy group), and glucosinolates (glucoraphanin, neoglucobrassicin) are present in higher amounts in SVT and EVT compared to HS, which, on the contrary, showed a higher content of glucose. In plants, acetate primarily functions in its activated form as acetyl-coenzyme A (Acetyl-CoA) [43]. It plays a central role in energy metabolism, storage, and utilization through the citric acid cycle (also known as the Krebs cycle or TCA cycle). Furthermore, acetyl-CoA serves as a central hub linking lipid and carbohydrate metabolism, and secondary metabolites biosynthesis, including flavonoids [44]. The accumulation of glucosinolates and flavonoids in D. muralis leaves improved its organoleptic properties, such as spicy flavor [3]. Additionally, 2-oxoglutarate, an intermediate of the Krebs cycle, plays a key role in carbon and nitrogen metabolism in plants. It also acts as a cofactor for a diverse range of enzymes involved in amino acid, glucosinolate, flavonoid, and alkaloid metabolism [45].

A significant aspect was the elevated flavonoid content observed in both types of vermitea (EVT and SVT), with particular emphasis on methoxy-modified quercetin analogues. This was evidenced by a highly significant signal at 3.62 ppm in the NMR spectrum, corresponding to the methoxy group. The application of vermitea upregulated the O-methyltransferase gene expression in plants [40]. O-methyltransferase plays a crucial role in the biosynthesis and diversification of flavonoids by methylation of hydroxyl groups on flavonoid precursors [46]. Methylation is a modification that flavonoids undergo, which can alter their stability, reactivity, and solubility. Flavonoid methylation requires methyl donors (SAM: S-adenosylmethionine), which are linked to nitrogen metabolism [47]. Moderate stress, as the presence of organic inputs, can shift flavonoid metabolism from hydroxy (-OH) to methoxy (-OCH₃) derivatives to increase the antioxidant capacity or the stability against oxidation.

Organic fertilizers stimulate the production of shikimic acid, which leads to an increased production of phenolic compounds and flavonoids [48]. Vermitea provides bioactive compounds or induces stress responses that trigger the synthesis of protective metabolites such as flavonoids and glucosinolates, which are involved in plant defense and antioxidant activity. This is also confirmed by the higher amount of glucose found in Hoagland solution growth conditions, where there is a lack of stress that induces a metabolic shift, and the more stable carbohydrate metabolism is preferred. Indeed, HS provides a well-balanced supply of nutrients, leading to normal growth in D. muralis without the need for increased secondary metabolism, such as flavonoids and glucosinolates. Moreover, flavonoids are powerful antioxidants that contribute to human health by reducing oxidative stress and inflammation. Glucosinolates, including glucoraphanin and neoglucobrassicin, are known for their potential anticancer properties and their role in detoxification processes in the human body [49]. Since these compounds are more abundant in SVT and EVT, plants grown under these conditions could offer greater health benefits.

From a nutritional perspective, SVT and EVT improve the nutrient profile of D. muralis compared to HS [14]. Brassica vegetables are a rich source of phenolic compounds, characterized by the presence of flavonoids and glucosinolates, which impact the flavor and acceptance of food and have potential health benefits [50]. Flavonoids, low molecular weight secondary metabolites located in cell vacuoles, play key roles in plant defense mechanisms. Several studies have explored the chemoprotective characteristics of rocket plants and have found that their flavonoid contents are induced by different light levels during the growth period [51,52,53]. In the present study, the environmental conditions were kept consistent for all treatments, so the observed variation in metabolites is not influenced by light factors. Therefore, the accumulation of phenolic compounds results also greatly influenced by biotic and abiotic stress, as well as nutrient availability [54].

Vermitea is rich in nitrogen, phosphorus, and potassium (NPK), as well as trace elements such as iron (Fe), zinc (Zn), and copper (Cu). The PLS-DA analysis performed here showed an interesting correlation between specific metabolites and metal contents, including differences in the absorption of these metals as previously discussed [14]. The higher levels of copper (Cu) and zinc (Zn) significantly modulated the phenol and flavonoid contents [55,56,57]. The enhanced content of Cu and Zn is closely related to higher methoxy flavonoids in EVT, whereas the P and K contents are associated with higher glucose in HS growth conditions. This effect may be due to the role of both P and K in various metabolic processes that influence sugar production and utilization [58]. Furthermore, an adequate phosphorus supply enhanced the activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a central enzyme in the Calvin–Benson cycle, thereby promoting photosynthetic efficiency and subsequently increasing glucose accumulation [59].

Another modification in the metabolism of plants grown in vermitea (EVT and SVT) is related to certain amino acids, particularly phenylalanine, which is converted into 4-coumaroyl-CoA by the phenylpropanoid pathway, producing flavonoids [60]. Amino acids contribute to the accumulation of osmolytes, thus playing a key role in plant stress tolerance mechanisms [61]. The amino acids present in plant physiological structures serve as fundamental precursors for protein synthesis, which supports plants in metabolism regulation and stress adaptation [62]. Additionally, various metabolites differentiating EVT and SVT treatments were also identified. The plants grown in SVT accumulated higher levels of amino acids, including leucine and citrate, compared to those grown in HS. This is because the vermitea contains organic nitrogen released by organic matter decomposition, which contributes to the increased amino acid profile.

Focusing on amino acids such as leucine, which is a branched-chain amino acid (BCAA), it plays a significant role in plant energy metabolism. Leucine was found in higher content in vermitea compared to HS, particularly in SVT samples. The carbon skeleton of BCAA is converted to the intermediates of the Krebs cycle for ATP production [63]. Additionally, citrate is the 1st intermediate of the Krebs cycle and is critical for various metabolic networks involved in the production of amino acids and phytohormones. Citrate also induces antioxidant defense systems, increases chlorophyll content and influences secondary metabolism to improve plant growth even under stress [64].

These findings are also supported by the referenced literature [65,66,67], indicating a higher accumulation of amino acids in plants treated with vermicompost.

5. Conclusions

The present study elucidated the influence of organic nutrient solutions, specifically SVT and EVT, in comparison to the conventional HS, on metabolite differentiation in D. muralis leaves. Plants cultivated in SVT and EVT systems accumulated higher concentrations of phenolic compounds compared to those grown in HS. Both organic nutrient solutions (SVT and EVT) enhanced the accumulation of acetate, alanine, and 2-oxoglutarate, along with key secondary metabolites including flavonoids and glucosinolates. Furthermore, SVT exhibited a higher level of leucine and citrate and a lower accumulation of glucose in comparison to HS. Notably, the increased accumulation of phenolic compounds in plants treated with EVT and SVT not only enriched the nutritional and nutraceutical value of D. muralis leaves but also improved sensory attributes such as aroma and taste. However, higher crop yield was observed in EVT and HS treatments relative to SVT. Therefore, the use of EVT as an organic hydroponic solution appears to be more beneficial for achieving higher yields along with improved nutritional quality and antioxidant contents. This approach may contribute to enhancing farmers’ economic returns while promoting better health among consumers. Moreover, the variations in the metabolite profile constitute a criterion for selecting the appropriate growth media to produce rocket plants with improved nutraceutical characteristics. In this context, the EVT system is recommended, as it supports improved plant growth alongside elevated levels of nutrients and flavonoids, key antioxidant compounds.

Future research should aim to identify the microbial communities present in vermicompost tea to uncover the key plant–microbe interactions responsible for the observed metabolic shifts in treated plants. Integrating metagenomic and metatranscriptomic approaches will be essential to elucidate these complex mechanisms. In parallel, transcriptomic analyses are recommended to clarify the molecular pathways driving the enhanced flavonoid production induced by organic nutrient solutions.