Romanian Wild-Growing Armoracia rusticana L.—Untargeted Low-Molecular Metabolomic Approach to a Potential Antitumoral Phyto-Carrier System Based on Kaolinite

Horseradish is a globally well-known and appreciated medicinal and aromatic plant. The health benefits of this plant have been appreciated in traditional European medicine since ancient times. Various studies have investigated the remarkable phytotherapeutic properties of horseradish and its aromatic profile. However, relatively few studies have been conducted on Romanian horseradish, and they mainly refer to the ethnomedicinal or dietary uses of the plant. This study reports the first complete low-molecular-weight metabolite profile of Romanian wild-grown horseradish. A total of ninety metabolites were identified in mass spectra (MS)-positive mode from nine secondary metabolite categories (glucosilates, fatty acids, isothiocyanates, amino acids, phenolic acids, flavonoids, terpenoids, coumarins, and miscellaneous). In addition, the biological activity of each class of phytoconstituents was discussed. Furthermore, the development of a simple target phyto-carrier system that collectively exploits the bioactive properties of horseradish and kaolinite is reported. An extensive characterization (FT-IR, XRD, DLS, SEM, EDS, and zeta potential) was performed to investigate the morpho-structural properties of this new phyto-carrier system. The antioxidant activity was evaluated using a combination of three in vitro, non-competitive methods (total phenolic assay, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, and phosphomolybdate (total antioxidant capacity)). The antioxidant assessment indicated the stronger antioxidant properties of the new phyto-carrier system compared with its components (horseradish and kaolinite). The collective results are relevant to the theoretical development of novel antioxidant agent fields with potential applications on antitumoral therapeutic platforms.


Introduction
Armoracia rusticana G. Gaertn., B. Mey. & Scherb (Armoracia rusticana L.) from the Brassicaceae family has been part of traditional European medicine since ancient times. The first mention of the healing effects of this plant (analgesic, diuretic, and antiparasitic) occurs in De Materia Medica [1]. Dacian medicine recommends horseradish as an anti-inflammatory cure for colds, coughs, and migraines [1]. Currently, horseradish root is used globally and on a large scale in food, food preservation, and traditional medicine [1]. phenol reagent (2 N), potassium persulfate, sodium phosphate, ammonium molybdate, and potassium chloride of 99% purity or higher were purchased from Sigma-Aldrich (München, Germany). Propyl gallate (purum) was purchased from Fluka (Buchs, Switzerland). The horseradish sample (leaves (28 cm in height) and roots (lengths of about 35 cm) were collected in November 2022 from the area of Timis County, Romania (geographic coordinates 45 • 45 59.99" N 21 • 17 60.00" E) and taxonomically authenticated at the University of Medicine and Pharmacy Craiova, Romania. Kaolinite was purchased from local market in Timisoara, Romania. The double distilled water (DDW) was used throughout the experiments.

Plant-Sample Preparation for Chemical Screening
The plant samples (roots and leaves) were cut and then quickly frozen in liquid nitrogen (180 • C). Subsequently, they were ground and sieved to obtain a particle size lower than 0.45 mm and then stored at −38 • C to prevent enzyme-mediated degradation of phytoconstituents, in a 100 mL conical flask containing 1.5 g dried plant sample and 15 mL of solvent (methanol/chloroform = 1:1). Subsequently, the mixture was subjected to sonication extraction for 30 min at 35 • C with a frequency of 60 kHz. The resulting solution was concentrated using a rotary evaporator, and the obtained residue was dissolved in 10 mL MeOH. The obtained extract was centrifuged (10,000 rot/min, 10 min), and the supernatant was filtered through a 0.2 µm syringe filter and stored at −25 • C until further analysis. All samples were prepared in triplicate.

GC-MS Separation Conditions
The oven-temperature program started from 50 • C to 300 • C with a rate of 5 • C/minute, and it was finally kept at this temperature for 3 min. The temperature of the injector was 280 • C and the temperature at the interface was 230 • C. The compounds' mass was registered at 70 eV ionization energy starting after 3 min of solvent delay. The source of the mass spectrometer was heated at 235 • C and the MS quad was heated at 165 • C. The mass values of identified compounds were scanned from 50 amu to 570 amu. Compounds were identified based on their mass spectra, which were compared to the NIST0.2 mass-spectra-library database (USA National Institute of Science and Technology Software, (NIST, Gaithersburg, MD, USA). Furthermore, the calculated retention indices (RIs) for each compound were compared with the Adams indices in the literature (Table 1) [40].

Mass Spectrometry
The MS experiments were performed using EIS-QTOF-MS (Bruker Daltonics, Bremen, Germany). The mass spectra were acquired in the positive ion mode in a mass range of 100-3000 m/z, scan speed was 2.0 scans/s, collision energy was 25-85 eV, and the temperature of source block was 80 • C. The identification of phytoconstituents was based on standard library NIST/NBS-3 (National Institute of Standards and Technology/National Bureau of Standards) (NIST, Gaithersburg, MD, USA). The obtained mass-spectra values and the identified secondary metabolites are presented in Table 2.

Phyto-Carrier System Preparation
For each analysis, 2.5 g of sample was prepared from dried horseradish, and kaolinite powder was added (horseradish/kaolinite nanoparticles = 1:3) at room temperature (22 • C), ground, and homogenized for 10 min using a pestle and mortar.

Characterization of the Phyto-Carrier System 2.2.1. Fourier-Transform Infrared (FTIR) Spectroscopy
Data collection was conducted after 20 recordings at a resolution of 4 cm −1 , in the range of 4000-400 cm −1 , on Shimadzu AIM-9000 with ATR devices (Shimadzu, Kyoto, Japan).

XDR Spectroscopy
The X-ray powder diffraction (XRD) was performed using a Bruker AXS D8-Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) equipped with a rotating sample stage, Anton Paar TTK low-temperature cell (−180 • C ÷ 450 • C), high vacuum, inert atmosphere, relative humidity control, and Anton Paar TTK high-temperature cell (up to 1600 • C). The XRD patterns were compared with those from the ICDD Powder Diffraction Database (ICDD file 04-015-9120). The average crystallite size and the phase content were determined using the whole-pattern profile-fitting method (WPPF).

Scanning-Electron Microscopy (SEM)
The SEM micrographs were obtained with a SEM-EDS system (QUANTA INSPECT F50) equipped with a field-emission gun (FEG), 1.2 nm resolution, and energy-dispersive X-ray spectrometer (EDS) with a MnK resolution of 133 eV.

Dynamic Light Scattering (DLS) Particle-Size-Distribution Analysis
The DLS analysis was carried on a Microtrac/Nanotrac 252 (Montgomeryville, PA, USA). Each sample was analyzed in triplicate at room temperature (22 • C) at a scattering angle of 172 • .

Determination of Total Phenolic Content
The total phenolic contents in the newly phyto-carrier system and horseradish samples were determined spectrophotometrically according to the Folin-Ciocalteu procedure adapted from the literature [41].
A volume of 2 mL of Folin-Ciocalteu reagent (0.2 N) and 0.2 mL of each sample were vortexed and stored at room temperature (22 • C) for 8 min, in the dark. Sequentially, 2 mL sodium carbonate (7.5%) was added. Next, after two h of incubation at room temperature (vortexed in the dark) the absorbance was measured at 725 nm using a Tecan i-control, 1.10.4.0 infinite 200 Pro spectrophotometer with Corning 96 flat-bottomed clear polystyrol plates (Tecan, Männedorf, Switzerland). The phenol content was expressed in gallic acid equivalents (mg GAE/g sample) using a propyl gallate standard calibration curve between 1 mg/mL and 12.5 µg/mL in methanol [42]. Sample extract concentrations were calculated based on the linear equation obtained from the standard curve (y = 0.9873x − 0.0989).

DPPH Radical-Scavenging Assay
The stock solution was prepared by dissolving 2 mg DPPH in 20 mL MeOH followed by dilutions for a calibration curve with a range of concentrations between 3.12 µg/mL and 0.1 mg/mL. Serial dilutions of ascorbic acid and β-carotene were used as positive standards and MeOH as a vehicle control sample. The ratio (v/v) of DPPH to samples was of 1:1. All samples were placed, in triplicate, in a 96-well plate and stored at 22 • C for 30 min in the dark. At 515 nm, the absorbance was determined on a Tecan i-control, 1.10.4.0 infinite 200 Pro spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland).
The obtained results were used to calculate the average and the inhibition percentage (Inh%) (Equation (1)). Inh% = (A0 − As)/A0 × 100 (1) where: A0 = vehicle control absorbance; As-sample absorbance. Further, the IC 50 value was obtained from the inhibition percentage using the equation of a calibration curve generated for each sample and standard. The results were presented as Inh% versus concentration (µg/mL) [43].

Phosphomolybdate Assay (Total Antioxidant Capacity)
The total-antioxidant-capacity assay of the new phyto-carrier system and horseradish samples was carried out by the phosphomolybdenum procedure using ascorbic acid as standard [44].
A volume of 5 mL reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) and 0.5 mL of each sample were placed into a water bath at 95 • C for 120 min. Next, the mixed solutions were cooled at room temperature (22 • C). The absorbance was measured at 765 nm using a UV-VIS Perkin-Elmer Lambda 35 (Perkin Elmer, Waltham, MA, USA).
A blank solution was used (5 mL reagent was added in 0.5 mL methanol, and then the mixture was incubated in the same experimental conditions (at 95 • C for 120 min, and then cooled at room temperature (22 • C)). Total antioxidant capacity was determined according to the following equation (Equation (2)) Total antioxidant capacity (%) = [(Abs. of control − Abs. of sample)/(Abs. of control] × 100 (2) The results are presented as µg/mL of ascorbic acid equivalents (AAE).

Statistical Analysis
All results were obtained with Microsoft Office Excel 2019. Data were used to calculate the average of three replicates for all samples, and all calibration curves and concentrations.

Results and Discussion
Plants contain an extensive range of categories of secondary metabolites, with complex chemical compositions [45,46].
Nevertheless, a specific plant's biological activity is the synergistic action of whole phytoconstituent result. Furthermore, researchers have reported that various biotic or abiotic factors (stress, pathogens, and others) altered the metabolite balance and, implicitly, their variability and interrelation [56][57][58]. In addition, several other elements (the part of the plant used, the extraction process, and the solvent used) influence the type and pro-portion of bioactive compounds collected from plants [58][59][60][61]. Therefore, a plant extract's pharmacological activity differs from the experimental conditions, making it difficult to evaluate the relationship between chemical composition and therapeutic effect [58].
The chemical screening of the phytoconstituents from the horseradish sample was carried out via gas chromatography coupled with mass spectroscopy (GC-MS) and electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) analysis.
The gas-chromatography method coupled with mass spectroscopy (GC-MS) is the most convenient technique for secondary metabolites with relatively low molecular mass (volatile compounds, fatty acids, etc.), providing efficient separation and identification [62].
The GC-MS analysis ( Figure 1) revealed the separation of several low-molecularweight metabolites from the horseradish sample.
Nevertheless, a specific plant's biological activity is the synergistic action of whole phytoconstituent result. Furthermore, researchers have reported that various biotic or abiotic factors (stress, pathogens, and others) altered the metabolite balance and, implicitly, their variability and interrelation [56][57][58]. In addition, several other elements (the part of the plant used, the extraction process, and the solvent used) influence the type and proportion of bioactive compounds collected from plants [58][59][60][61]. Therefore, a plant extract's pharmacological activity differs from the experimental conditions, making it difficult to evaluate the relationship between chemical composition and therapeutic effect [58].
The chemical screening of the phytoconstituents from the horseradish sample was carried out via gas chromatography coupled with mass spectroscopy (GC-MS) and electrospray ionization-quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) analysis.
The gas-chromatography method coupled with mass spectroscopy (GC-MS) is the most convenient technique for secondary metabolites with relatively low molecular mass (volatile compounds, fatty acids, etc.), providing efficient separation and identification [62].
The GC-MS analysis ( Figure 1) revealed the separation of several low-molecular-weight metabolites from the horseradish sample. The results are summarized in Table 1, which presents the tentative compound identification from the horseradish sample using GC-MS. The results are summarized in Table 1, which presents the tentative compound identification from the horseradish sample using GC-MS. RI-retention indices calculated based upon a calibration curve of a C8-C20 alkane standard mixture.
The GC-MS analysis showed the presence of seventeen major components, accounting for 95.13% of the total peak area in the horseradish samples ( Figure 1).
However, thermally unstable biomolecules require additional procedures (for instance, derivatization). Therefore, the mass-spectrometry method was selected for the metaboliteprofile screening [65].

Mass-Spectrometric Analysis of Horseradish Sample
The spectra revealed a complex combination of low-molecular-weight components, of which some were detected. The mass spectra of the identified metabolites were compared with those of the NIST/EPA/NIH Mass Spectral Library 3.0 database, in addition to a literature review [7,48,55,66]. The mass spectrum and the phytoconstituents identified by the ESI-QTOF-MS analysis are presented in Figure 2 and Table 2, respectively.        The metabolite profile from the horseradish sample conducted through the GC-MS and mass spectroscopy corroborated the data reported in the literature [6][7][8][48][49][50][51][52][53][54][55][63][64][65].

Screening and Classification of the Differential Metabolites
The 90 secondary metabolites identified through mass spectroscopy were assigned to different chemical classes: glucosilates (18.9%), fatty acids (11.12%), isothiocyanates (8.9%), amino acids (8.9%), phenolic acids (6.67%), flavonoids (4.45%), terpenoids (3.34%), coumarins (2.23%), and miscellaneous. The assignment of the identified secondary metabolites into different chemical categories is presented in Table 3.     Figure 3 presents the classification chart of the phytoconstituents from the horseradish sample based on the data analysis reported in Table 3. miscellaneous naphthalene 3-phenylpropionitrile 2-sec-butyl-3 methoxypyrazine Figure 3 presents the classification chart of the phytoconstituents from the horseradish sample based on the data analysis reported in Table 3. According to Figure 3, glucosinolates are the largest category of phytochemicals, comprising about 19% of the total found in the horseradish sample. Recent studies demonstrated their antioxidant, anti-inflammatory, and antitumoral properties [7,66,67].
Isothiocyanates are a category of metabolites characteristic of cruciferous plants, with remarkable anti-cancer, anti-inflammatory, and neuroprotective effects [67,68].
Organo-sulfur phytoconstituents represented over 30% of all the metabolites identified in the horseradish sample. Various studies have shown that sulfur phytochemicals possess antioxidant, antiviral, antifungal, antibacterial, and antitumor properties [7,54,69,70]. According to Figure 3, glucosinolates are the largest category of phytochemicals, comprising about 19% of the total found in the horseradish sample. Recent studies demonstrated their antioxidant, anti-inflammatory, and antitumoral properties [7,66,67].
Isothiocyanates are a category of metabolites characteristic of cruciferous plants, with remarkable anti-cancer, anti-inflammatory, and neuroprotective effects [67,68].
Fatty acids represented more than 11% of the phytoconstituents identified in the horseradish sample. These secondary metabolites exhibit antioxidant, anti-inflammatory, cardio, and neuroprotective activities [71,72].
Flavonoids are other category of secondary metabolites identified in the horseradish sample with notable pharmacological proprieties, including antioxidant, anti-inflammatory, antitumoral, and antimicrobial properties, as well as activities against neurodegenerative diseases (Alzheimer's) [73,79,83].
The aromatic compounds of volatile metabolites (VOCs) identified in the horseradish sample are shown in Table 4 and Figure 4.    The predominant aromatic components of the investigated Romanian horseradish depend on different conditions (climatic conditions, maturity soil parameters, varieties, harvest time, and others) [4,7,[47][48][49].

Phyto-Carrier System
The main challenges in the novel therapeutic approaches to cancer are the drug resistance of cancer cells, determined by the reduced retention interval, low permeability, the triggering of inactivation by the immune system, and the lack of specificity [89,90].
Hence, the development of an innovative phyto-carrier target system with cumulative and synergistic kaolinite and horseradish biological activity could make it possible to overcome the limitations related to vectorization, site-specific distribution, prolonged release, and membrane permeability.

FT-IR Spectroscopy
The use of FTIR is one of the most common analytical techniques, and it is considered fundamental in the analysis of complex carrier systems due to its features (sensitivity, flexibility, robustness, and specificity), allowing the investigation of interactions between biomolecules and mineral components [90].
The incorporation of the horseradish phytoconstituents into the pores of the kaolinite particles was successfully achieved and confirmed through FT-IR spectroscopy. Figure 5A presents the spectra of the horseradish, the kaolinite particles, and the new phyto-carrier system. Their fragrances are unique, encompassing a purgent aroma with rocket and sulfuric, green, sweet-vanilla, and floral notes [4,7,47,48].

Phyto-Carrier System
The main challenges in the novel therapeutic approaches to cancer are the drug resistance of cancer cells, determined by the reduced retention interval, low permeability, the triggering of inactivation by the immune system, and the lack of specificity [89,90].
Hence, the development of an innovative phyto-carrier target system with cumulative and synergistic kaolinite and horseradish biological activity could make it possible to overcome the limitations related to vectorization, site-specific distribution, prolonged release, and membrane permeability.

FT-IR Spectroscopy
The use of FTIR is one of the most common analytical techniques, and it is considered fundamental in the analysis of complex carrier systems due to its features (sensitivity, flexibility, robustness, and specificity), allowing the investigation of interactions between biomolecules and mineral components [90].
The incorporation of the horseradish phytoconstituents into the pores of the kaolinite particles was successfully achieved and confirmed through FT-IR spectroscopy. Figure  5A presents the spectra of the horseradish, the kaolinite particles, and the new phyto-carrier system.
The data obtained and presented in Figure 5 confirm the successful development of the new phyto-carrier system.

X-ray-Diffraction Spectroscopy
The XRD technique was used to obtain information about the atomic structure of the phyto-carrier system and the raw materials. Figure 6 displays the XRD patterns of the horseradish sample and the new phytocarrier system.

X-ray-Diffraction Spectroscopy
The XRD technique was used to obtain information about the atomic structure of the phyto-carrier system and the raw materials. Figure 6 displays the XRD patterns of the horseradish sample and the new phytocarrier system.  In the XRD spectrum of the new phyto-carrier system, the characteristic XRD peaks of the kaolinite and horseradish samples are easily observable. Hence, the absorption peaks at 2θ (degrees) values of 12 • , 25 • , 34 • , 36 • , and 51 • can be assigned to a triclinic structure [105].
The XRD pattern of the horseradish sample ( Figure 6) was in the range of 11.8-34.6 • , with large bands and weak peaks characteristic of amorphous phases, which can be attributed to the phytoconstituents from the horseradish (minerals, hydroxides, and fibers).

Scanning-Electron Microscopy-Energy-Dispersive X-ray (SEM-EDX)
Scanning-electron microscopy-energy-dispersive X-ray (SEM-EDX) is a versatile technique to investigate the morphologies, compositions, and microstructures of materials. In some complex materials, it allows the identification of the component phases through qualitative chemical analysis [106].
The morphological changes (the size, shape, and distribution of the particles) in the horseradish and kaolinite samples before and after the preparation of the new phyto-carrier system were investigated by using the SEM-EDX technique.
To acquire insights, the SEM micrographs were recorded at different magnifications. The obtained two-dimensional images are shown in Figure 7. als. In some complex materials, it allows the identification of the component phases through qualitative chemical analysis [106].
The morphological changes (the size, shape, and distribution of the particles) in the horseradish and kaolinite samples before and after the preparation of the new phyto-carrier system were investigated by using the SEM-EDX technique.
To acquire insights, the SEM micrographs were recorded at different magnifications. The obtained two-dimensional images are shown in Figure 7. The SEM micrograph of the kaolinite sample ( Figures 7A,B) exhibited a heterogeneous size distribution of small anhedral and pseudo-hexagonal particles up to 5 μm in size [25].
It appears that the horseradish micrographs ( Figures 7C,D) indicated the presence of a heterogeneous fibrous structure, with a thickness of about a few μm, with porous regions with irregular shapes. These porous regions allowed the arrest of the kaolinite particles.
The morphology of the phyto-carrier system ( Figure 7E,F) indicated the presence of kaolinite particles both on the surface and in the porous areas of the horseradish sample. Changes in the sizes of the horseradish and kaolinite particles (reduction) were observed, which can be explained by the experimental conditions of the new phyto-carrier system preparation.
Accompanying the SEM spectra are EDX analyses on the elemental composition of the kaolinite and phyto-carrier investigated ( Figure 8A,B). The SEM micrograph of the kaolinite sample ( Figure 7A,B) exhibited a heterogeneous size distribution of small anhedral and pseudo-hexagonal particles up to 5 µm in size [25].
It appears that the horseradish micrographs ( Figure 7C,D) indicated the presence of a heterogeneous fibrous structure, with a thickness of about a few µm, with porous regions with irregular shapes. These porous regions allowed the arrest of the kaolinite particles.
The morphology of the phyto-carrier system ( Figure 7E,F) indicated the presence of kaolinite particles both on the surface and in the porous areas of the horseradish sample. Changes in the sizes of the horseradish and kaolinite particles (reduction) were observed, which can be explained by the experimental conditions of the new phyto-carrier system preparation.
Accompanying the SEM spectra are EDX analyses on the elemental composition of the kaolinite and phyto-carrier investigated ( Figure 8A,B).

particles.
The morphology of the phyto-carrier system ( Figure 7E,F) indicated the presence of kaolinite particles both on the surface and in the porous areas of the horseradish sample. Changes in the sizes of the horseradish and kaolinite particles (reduction) were observed, which can be explained by the experimental conditions of the new phyto-carrier system preparation.
Accompanying the SEM spectra are EDX analyses on the elemental composition of the kaolinite and phyto-carrier investigated ( Figure 8A According to the data from the EDX (Figure 7A), the predominant element contents in the kaolinite sample were silica, aluminum, magnesium, calcium, potassium, iron, sodium, oxygen, and sulphur. Overall, the chemical analysis revealed the significant oxides SiO2, Al2O3, Fe2O3, MgO, CaO, Na2O, K2O5, and SO −3 , which was in good agreement with the data reported in the literature [25].
The comparative analysis in Figure 8B highlights the presence of peaks corresponding to the kaolinite ( Figure 8A) in the new phyto-carrier system. The EDX results confirmed the preparation of the new phyto-carrier system.

Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) is a fast and very efficient method for determining the sizes of particles and the particle-size distribution (PSD) in suspensions [107]. Particle-size measurement is established indirectly by using the intensity of the light-scattered fluctuations, yielding the rate of the Brownian motion [107].
The DLS method was used to obtain information about the average mean particle size of the phyto-carrier system and its raw components. The DLS results are displayed in Figure 9. According to the data from the EDX (Figure 7A), the predominant element contents in the kaolinite sample were silica, aluminum, magnesium, calcium, potassium, iron, sodium, oxygen, and sulphur. Overall, the chemical analysis revealed the significant oxides SiO 2 , Al 2 O 3 , Fe 2 O 3 , MgO, CaO, Na 2 O, K 2 O 5 , and SO −3 , which was in good agreement with the data reported in the literature [25].
The comparative analysis in Figure 8B highlights the presence of peaks corresponding to the kaolinite ( Figure 8A) in the new phyto-carrier system. The EDX results confirmed the preparation of the new phyto-carrier system.

Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) is a fast and very efficient method for determining the sizes of particles and the particle-size distribution (PSD) in suspensions [107]. Particle-size measurement is established indirectly by using the intensity of the light-scattered fluctuations, yielding the rate of the Brownian motion [107].
The DLS method was used to obtain information about the average mean particle size of the phyto-carrier system and its raw components. The DLS results are displayed in Figure 9.
The comparative analysis in Figure 8B highlights the presence of peaks corresponding to the kaolinite ( Figure 8A) in the new phyto-carrier system. The EDX results confirmed the preparation of the new phyto-carrier system.

Dynamic Light Scattering (DLS)
Dynamic light scattering (DLS) is a fast and very efficient method for determining the sizes of particles and the particle-size distribution (PSD) in suspensions [107]. Particle-size measurement is established indirectly by using the intensity of the light-scattered fluctuations, yielding the rate of the Brownian motion [107].
The DLS method was used to obtain information about the average mean particle size of the phyto-carrier system and its raw components. The DLS results are displayed in Figure 9. The average diameter size of the kaolinite particles was 500.03 nm ( Figure 9A), corroborating the SEM results. In the horseradish sample ( Figure 9B), the average diameter of the particles was 100.2 nm.
The DLS pattern of the new phyto-carrier system ( Figure 9C) exhibited two peaks that can be attributed to the kaolinite and horseradish particles, distributed in a narrow range. The mean diameter of the kaolinite in the phyto-carrier system was 277.5 nm. The second mean of the hydrodynamic diameter, associated with the horseradish particles, was about 186.4 nm. The fact that the average diameter of the horseradish particles in the phyto-carrier increased compared to that determined in the horseradish sample can be attributed to the loading of the pores on the plant surfaces with the kaolinite particles, which was confirmed by the results of the SEM analysis.
Furthermore, the reduction in the mean size of the kaolinite particles from 500.03 nm ( Figure 9A) to 277.5 nm ( Figure 9C) was attributed to the experimental conditions for the preparation of the new phyto-carrier. In addition, Figure 9C shows well-dispersed parti- The average diameter size of the kaolinite particles was 500.03 nm ( Figure 9A), corroborating the SEM results. In the horseradish sample ( Figure 9B), the average diameter of the particles was 100.2 nm.
The DLS pattern of the new phyto-carrier system ( Figure 9C) exhibited two peaks that can be attributed to the kaolinite and horseradish particles, distributed in a narrow range. The mean diameter of the kaolinite in the phyto-carrier system was 277.5 nm. The second mean of the hydrodynamic diameter, associated with the horseradish particles, was about 186.4 nm. The fact that the average diameter of the horseradish particles in the phyto-carrier increased compared to that determined in the horseradish sample can be attributed to the loading of the pores on the plant surfaces with the kaolinite particles, which was confirmed by the results of the SEM analysis.
Furthermore, the reduction in the mean size of the kaolinite particles from 500.03 nm ( Figure 9A) to 277.5 nm ( Figure 9C) was attributed to the experimental conditions for the preparation of the new phyto-carrier. In addition, Figure 9C shows well-dispersed particles of horseradish and kaolinite, which indicates the high stability of the new phyto-carrier system.

Zeta Potential
The zeta-potential method determines the charge of a particle in a suspension, providing an estimation of interactions between particles and the suspension stability.
The zeta-potential value of the kaolinite particles was −35.09 mV, indicating the high stability of the suspension, in good agreement with the data reported in the literature [108].
The zeta potential changed to −23.12 mV for the phyto-carrier system, indicating high biocompatibility.

Screening of Antioxidant Activity
For a specific herb, the total antioxidant capacity (TAC) is the outcome of the cumulative action of entire antioxidant classes from its composition [37]. The adequate investigation of the antioxidant activity of a plant requires an appropriate variety of tests to address the mechanism of action characteristic of each category of phytochemicals [37,38,109,110].
Various chemical (spectrometric, chromatographic, and electrochemical) and biochemical methods have been developed for the assessment of the antioxidant capacities of different biomolecules [37,38,109,110]. The most common are the in vitro tests, divided based on the reaction-mechanism type into hydrogen-atom transfer (HAT) and electron transfer (ET) methods [37,38,109,110].
The biological activity of a plant varies depending on the complexity of the chemical composition and, implicitly, on the collective, complementary, and the synergistic actions of a variety of secondary metabolites. Moreover, the antioxidant activities of plants differ, depending on morphological parts, degree of maturity, and exogenous parameters (temperature, pH, humidity, and others) [37].
Hence, the antioxidant activity of the phyto-carrier system is a combined result of the complementary and synergistic actions of its components (horseradish and kaolinite). A total amount of ninety secondary metabolites from nine different chemical classes were identified in the horseradish sample. Consequently, to consider the antioxidant properties of the new phyto-carrier system more precisely, three different in vitro, noncompetitive methods were used (DPPH, Folin-Ciocalteu, and phosphomolybdate (total antioxidant capacity).
Hence, the antioxidant activity of the new phyto-carrier system and its components were evaluated in relation to the antioxidant standards of β-carotene and ascorbic acid. It is noteworthy that different studies reported the presence of β-carotene and ascorbic acid in the chemical composition of horseradish [114,115]. The data obtained are presented in Table 6 and Figure 10.  The obtained IC50 values indicated that the antioxidant activity of the new phyto-carrier system was higher than that of the horseradish sample, the kaolinite, and the ascorbic acid. For the new phyto-carrier system, the IC50 value was about half that of the horseradish sample. The increase in the antioxidant activity of the phyto-carrier system compared to the horseradish and kaolinite was in good agreement with the literature data [116,117]. The IC50 value for the beta-carotene standard can be explained by the experimental conditions (the low solubility of beta carotene in methanol) [118].

Folin-Ciocalteu Assay
This assay is widely used as a fast, simple, precise, and inexpensive measure of total phenolics from natural products based on an oxidation/reduction-reaction mechanism (electron transfer) [38,39,41,119,120].
The total polyphenolic contents (TPCs) of the horseradish sample and phyto-carrier system were determined and the obtained results are presented in Table 7. Table 7. Total polyphenolic contents in horseradish and the phytocarrier system.

Sample Name
Total Phenolic Content (µg/mL) horseradish 13.79667 phyto-carrier system 35.18658 According to the results, the total polyphenolic content identified in the new phyto-carrier system was more than 39% higher than that of the horseradish sample. The higher antioxidant capacity of the phyto-carrier system compared to the horseradish sample can be attributed to the synergistic action of the kaolinite and corresponds to the data reported in the literature [121]. The obtained IC50 values indicated that the antioxidant activity of the new phytocarrier system was higher than that of the horseradish sample, the kaolinite, and the ascorbic acid. For the new phyto-carrier system, the IC50 value was about half that of the horseradish sample. The increase in the antioxidant activity of the phyto-carrier system compared to the horseradish and kaolinite was in good agreement with the literature data [116,117]. The IC50 value for the beta-carotene standard can be explained by the experimental conditions (the low solubility of beta carotene in methanol) [118].

Folin-Ciocalteu Assay
This assay is widely used as a fast, simple, precise, and inexpensive measure of total phenolics from natural products based on an oxidation/reduction-reaction mechanism (electron transfer) [38,39,41,119,120].
The total polyphenolic contents (TPCs) of the horseradish sample and phyto-carrier system were determined and the obtained results are presented in Table 7. Table 7. Total polyphenolic contents in horseradish and the phytocarrier system.

Sample Name
Total Phenolic Content (µg/mL) horseradish 13.79667 phyto-carrier system 35.18658 According to the results, the total polyphenolic content identified in the new phytocarrier system was more than 39% higher than that of the horseradish sample. The higher antioxidant capacity of the phyto-carrier system compared to the horseradish sample can be attributed to the synergistic action of the kaolinite and corresponds to the data reported in the literature [121].
3.9.3. Phosphomolybdate Assay (Total Antioxidant Capacity) Phosphomolybdate (total antioxidant capacity) is a frequently used and precise assay used to evaluate the total antioxidant potentials of plant extracts or other complex mixtures of biomolecules. It is based on the Mo(VI)-to-Mo(V) reduction of the presence of antioxidants [44].
The phosphomolybdate assay (total antioxidant capacity) was used to determine the total antioxidant potential of the prepared phyto-carrier system compared to those of the horseradish and ascorbic acid. The obtained experimental results are displayed in Table 8 and Figure 11.  Sample Name horseradish 171.82 ± 0.00343 phyto-carrier system 248.96 ± 0.014 Figure 11. Graphic representation of phosphomolybdate (total antioxidant capacity) results expressed as µ g/mL AAE.
The phyto-carrier system displayed a higher antioxidant activity than the horseradish sample. This result can be attributed to the synergistic and complementary action of the phytoconstituents in the horseradish and the antioxidant mechanism of the kaolinite [28]. In addition, the kaolinite potentiated the antioxidant activities of secondary metabolites in the horseradish sample [121].

Conclusions
In this study, a new phyto-carrier system with particular morpho-structural properties and high antioxidant activity was prepared. The low-molecular-mass-metabolite profiling and the VOC-aroma profile of the Armoracia rusticana grown in the wild in Romania were determined. The biological activities of each identified phytoconstituent category in the horseradish were discussed. The development of the horseradish-kaolinite carrier system was confirmed through FTIR, EDX, XRD, DLS, zeta-potential, and SEM studies. The size distributions of the kaolinite and horseradish particles were investigated through a DSL analysis. The kaolinite and the phyto-carrier system's stability levels in aqueous suspensions were determined using a zeta-potential analysis. A combination of assays (DPPH, Folin-Ciocalteu, and phosphomolybdate (total antioxidant capacity)) was used to evaluate the antioxidant properties of the proposed phyto-carrier system. The results demonstrated the significantly higher antioxidant activity of the phyto-carrier compared with its components (horseradish and kaolinite). The phyto-carrier system displayed a higher antioxidant activity than the horseradish sample. This result can be attributed to the synergistic and complementary action of the phytoconstituents in the horseradish and the antioxidant mechanism of the kaolinite [28]. In addition, the kaolinite potentiated the antioxidant activities of secondary metabolites in the horseradish sample [121].

Conclusions
In this study, a new phyto-carrier system with particular morpho-structural properties and high antioxidant activity was prepared. The low-molecular-mass-metabolite profiling and the VOC-aroma profile of the Armoracia rusticana grown in the wild in Romania were determined. The biological activities of each identified phytoconstituent category in the horseradish were discussed. The development of the horseradish-kaolinite carrier system was confirmed through FTIR, EDX, XRD, DLS, zeta-potential, and SEM studies. The size distributions of the kaolinite and horseradish particles were investigated through a DSL analysis. The kaolinite and the phyto-carrier system's stability levels in aqueous suspensions were determined using a zeta-potential analysis. A combination of assays (DPPH, Folin-Ciocalteu, and phosphomolybdate (total antioxidant capacity)) was used to evaluate the antioxidant properties of the proposed phyto-carrier system. The results demonstrated the significantly higher antioxidant activity of the phyto-carrier compared with its components (horseradish and kaolinite). However, further studies are required to investigate the biological activity, bioavailability, and biocompatibility of the new phytocarrier system. This study may motivate future research on therapies in the area of advanced antitumoral agents.