Flavonoid and Phenolic Acids Content and In Vitro Study of the Potential Anti-Aging Properties of Eutrema japonicum (Miq.) Koidz Cultivated in Wasabi Farm Poland

Skin aging is a natural, unavoidable, and complex process caused by oxidative stress. As a consequence, it leads to an increase in the activation of extracellular matrix disruption enzymes and DNA damage. The search for natural sources that inhibit these mechanisms can be a good approach to prevent skin aging. The purpose of our study was to evaluate the composition of flavonoids and phenolic acids in the extracts obtained from the flowers, roots, and leaves of Eutrema japonicum cultivated in Poland. Then, the resultant extracts were subjected to an assessment of antioxidant, anti-collagenase, anti-elastase, anti-hyaluronidase, antibacterial, and cytotoxic properties. It was demonstrated that the extract from the flowers had the highest content of flavonoid glycosides (17.15 mg/g DE). This extract showed the greatest antioxidant, anti-collagenase, anti-elastase, and anti-hyaluronidase activities compared to the other samples. Importantly, the collagenase inhibitory activity of this extract (93.34% ± 0.77%) was better than that of positive control epigallocatechin gallate (88.49% ± 0.45%). An undeniable advantage of this extract was also to possess moderate antibacterial properties and no cytotoxicity towards normal human skin fibroblasts. Our results suggest that extracts from E. japonicum flowers may be considered as a promising antiaging compound for applications in cosmetic formulations.


Introduction
The plant kingdom is a valuable source of polyphenols, such as flavonoids, which exhibit beneficial antioxidant, antibacterial, antiaging, antiviral, antifungal, and antiinflammatory effects [1]. Thus, the use of plant extracts in cosmetic formulations has been gaining popularity during the past few years. Their use in cosmetic products was found to be more favorable than the application of single biologically active compounds. The search of natural substances with health-promoting activities, such as antioxidant, anti-elastase, and anti-collagenase properties, as well as without cytotoxic effects on the skin, has gained increasing attention. An example is plants rich in polyphenols that have a protective role in many diseases, such as inflammation, cardiovascular illnesses, and cancers. For these reasons, they are used in the healthcare, food, and cosmetics industries [2]. Flavonoids and phenolic acids, in view of their ROS-scavenging activity and/or ability to chelate metals, are often used in cosmetic, especially antiaging, formulations [3].

Phytochemical Analysis
The total phenolic content (TPC) was determined using Folin-Ciocalteu reagent, and the results were estimated as gallic acid equivalents (GAE) per g of dry extract (DE) ( Table 1). Among the four extracts of E. japonicum, the extract from the flowers (WJA) had the highest phenolic content (187.79 ± 6.86 mg GAE/g DE), followed by annual leaves (WJC) (77.92 ± 4.82 mg GAE/g DE), biennial leaves (WJD) (54.93 ± 0.99 mg GAE/g DE), and roots (WJB) (31.55 ± 2.17 mg GAE/g DE). It is worth noting that the results obtained for the extracts of E. japonicum in our study were better compared to the data presented for the extracts derived from other Wasabi species. For instance, Kang et al. [22] demonstrated that TPC for n-hexane and water fractions of 80% ethanol extract of the leaves of Wasabi koreana were 13.29 ± 0.43 and 26.35 ± 0.35 mg GAE/g of extract, respectively. Kim et al. [23] found lower amounts of phenolic compounds in the water extracts of the leaves (approx. 0.13 mg GAE/g) and of the roots (approx. 0.04 mg GAE/g) of W. koreana. Shin et al. [24] studied the total phenolic content in different organs of wasabi grown in an organic system. They showed that the total phenolic values in the methanol extract or in the boiled water extract of the roots ranged from 5.10 to 7.78 mg of tannic acid equivalent (TAE)/g of the dry weight (dw.) and from 3.40 to 4.64 mg TAE/g dw., respectively. In turn, the methanol extracts of the flowers and leaves possessed phenolic contents equal to 36.44 and 32.01 mg TAE/g dw., respectively. Thus, the results obtained in our study indicated that E. japonicum is a rich source of phenolic compounds. Table 1. The total content of phenolic (TPC), flavonoid (TFC), and phenolic acids (TPAC) in the flowers (WJA), roots (WJB), and leaves (annual-WJC and biennial-WJD) of E. japonicum. DE-dry extract; GAE-Gallic Acid Equivalent; CAE-Caffeic Acid Equivalent; QE-Quercetin Equivalent. Values were presented as the mean ± standard deviation (n = 9). The total flavonoid content of the different parts of E. japonicum cultivated in Poland was estimated by the previously described colorimetric method [25]. The data were expressed as quercetin equivalents (QE) per g of dry extracts (DE). The results presented in Table 1 showed that, among the examined samples, the flowers (WJA) exhibited the highest content of total flavonoids (45.08 ± 0.18 mg QE/g DE). A comparable content was noted for annual (WJC) and biennial (WJD) leaves (28.81 ± 0.11 and 29.77 ± 0.04 mg QE/g DE, respectively). The data obtained for the roots was five times lower compared to the value noted for the flowers (9.15 ± 0.10 mg QE/g DE). The results obtained in our study for the flowers and leaves were higher than those found by Shin et al. [24]. In their study, a quantitative estimation revealed that a methanol extract from the flowers of W. japonica (grown in an organic system) possessed a 11.52 mg QE/g dw. flavonoid content, followed by the leaves-3.25 mg QE/g dw. and fruits-0.64 mg QE/g dw. In the roots, petioles, and floral stalks, the flavonoid content was not detected. The higher amounts of flavonoids were found in the different extracts from the leaves of W. koreana (30.16 ± 0.37 to 104.16 ± 8.93 mg NE/g [22].

Sample
The total phenolic acids content (TPAC) in the studied extracts were presented in Table 1. The amounts ranged from 4.34 ± 0.08 to 17.20 ± 0.24 mg CAE/g DE. The highest TPAC content was observed for the flowers (WJA).
In the next step of our study, the phenolic acid and flavonoid compositions of the extracts obtained from E. japonicum were investigated using the LC-MS/MS method. The analysis was carried out using a previously validated and described method [26]. The results of the qualitative and quantitative analyses are presented in Table 2. The sample LC-ESI-MS/MS chromatogram (WJA) and mass spectra of the main compound isosaponarin are displayed in Figures S1 and S2, respectively.
Among the investigated parts of E. japonicum, the flowers (WJA) had the highest total content of phenolic acids and flavonoids. Only six phenolic acids (salicylic acid, p-coumaric acid, caffeic acid, ferulic acid, chlorogenic acid, and cis-sinapic acid) were identified in all the samples. Cis-sinapic acid was the most abundant phenolic acid in all the wasabi extracts (0.09 ± 0.0 to 0.77 ± 0.06 mg per g of DE). Chlorogenic acid was detected in a quantifiable amount in the extracts from the flowers (0.01 ± 0.01 mg/g DE), roots (0.62 ± 0.02 mg/g DE), and biennial leaves (0.42 ± 0.02 mg/g DE).
Therefore, our results were in good agreement with previous investigations on the identification of isosaponarin and ferulic acid in the leaves [11,20], isorhamnetin-3-glucoside and astragalin in the flowers [10], and trans-ferulic acid, as well as trans-pcoumaric acid, in the roots [27] of E. japonicum. To the best of our knowledge, the other phenolic acids and flavonoids were identified for the first time in the investigated species.

Skin-Related Activities
Skin aging is a natural, unavoidable, complex process caused by oxidative stress. As a result, an increase in the activation of extracellular matrix disruption enzymes and DNA damage was observed. Various intrinsic and extrinsic factors are responsible for this process, including genetic, hormonal, and metabolic changes, as well as exposure to environmental stress [28,29]. As various enzymes such as elastase, collagenase, or hyaluronidase are involved in the skin-aging process, plant extracts, as their inhibitors, are desirable. Thus, apart from the qualitative and quantitative analyses of flavonoids and phenolic acids, this study focused on skin-related activities of different parts of E. japonicum cultivated in Poland. In our research, we examined the in vitro antioxidant, anti-collagenase, anti-elastase, anti-hyaluronidase, antibacterial, and cytotoxic activities of different parts of E. japonicum.

Antioxidant Activity
The antioxidant activity was studied on a microplate scale in the cell-free systems. All the samples were studied in a concentration range from 10 to 150 µg/mL. It was demonstrated that all the investigated extracts exhibited the moderate scavenging capacity in a concentration-dependent manner (Table 3). For comparison, the radical scavenging activity of ascorbic acid (AA) was tested in the same conditions (IC 50 = 4.92 ± 0.32 µg/mL). The highest DPPH scavenging activity was shown for the extract from the flowers (WJA) (IC 50 = 28.72 ± 0.24 µg/mL), followed by annual (WJC) and biennial (WJD) leaves (IC 50 = 62.84 ± 0.08 and 69.10 ± 0.16 µg/mL, respectively). Table 3. The IC 50 values determined in the antioxidant tests. Data were expressed as the mean ± SD, n = 3. AA-ascorbic acid; Na 2 EDTA*2H 2 O-ethylenediaminetetraacetic acid, disodium dihydrate; nt-not tested. The flowers (WJA), roots (WJB), and leaves (annual-WJC and biennial-WJD) of E. japonicum. DPPH-2,2-diphenyl-1-picryl-hydrazyl radical; ABTS-2,2 -azino-bis-(3-ethylbenzothiazole-6-sulfonic acid; CHEL-metal chelating activity. * Statistically significant differences compared to AA; # statistically significant differences compared to Na 2 EDTA*2H 2 O, p < 0.05. The results of other published reports seem to be difficult to compare due to the other conditions used during the experiments. Nevertheless, antioxidant activity using the DDPH assay was studied for different fractions of 80% ethanol extract of the leaves of W. koreana [22]. The authors found that extracts of wasabi possessed low scavenging effects, with an IC 50 value ranging from 522.06 to 12,667.20 µg/mL (the IC 50 value for the positive control-ascorbic acid-was 41.93 µg/mL). Kim et al. [23] also studied the antioxidant capacity with a DPPH test of different parts of W. japonica. They found that the leaves and roots had IC 50 values of 7.64 ± 0.54 µg/mL and 16.95 ± 0.61 µg/mL, respectively. Eleven compounds isolated from the leaves of W. japonica were also evaluated by the DPPH radical scavenging assay. Ferulic acid, luteolin, isoorientin, and rutin showed a significant inhibitory activity. Importantly, luteolin (IC 50 = 4.09 ± 0.28 µg/mL) and isoorientin (IC 50 = 6.73 ± 0.45 µg/mL) showed a stronger activity compared to Trolox (IC 50 = 6.48 ± 0.73 µg/mL) [10].

Sample
Similar to the DPPH test, the ABTS •+ assay revealed that the extract from the flowers (WJA) possessed the strongest ability to scavenge free radicals (IC 50 = 11.68 ± 0.47 µg/mL), followed by WJD (IC 50 = 28.53 ± 0.72 µg/mL) and WJC (IC 50 = 33.25 ± 0.31 µg/mL). The ABTS •+ assay was also used to test the different extracts of W. koreana. Kang and coauthors found that different extracts of W. koreana possessed a low ABTS radical scavenging effect, with IC 50 values that ranged from 171.99 to 3103.04 µg/mL (the IC 50 for the positive control, ascorbic acid, was 39.07 µg/mL) [22].
It is commonly known that phenolic compounds may reduce oxidative stress by various mechanisms that depend on their chemical structures. One of them is the chelation of metal ions, such as iron, which plays a key role in the production of harmful oxygen species [30]. Under regular conditions, iron is stored and transported by ferritin or transferrin, which prevents the reaction of free iron ions with reactive oxygen species. The iron ions generate OH• radicals, which can react with lipids, causing their peroxidation [31]. Therefore, it is crucial to search for new natural compounds with the potential ability to chelate metal ions.
The chelating capacity was determined based on measurement of the percentage of inhibition of the formation of a ferrozine-Fe 2+ complex. The extracts from the flowers (WJA) and biennial (WJD) and annual (WJC) leaves possessed the activity of interfering with the formation of iron and ferrozine complexes, which suggests their high chelating capacity and ability to capture iron ions before ferrozine. The IC 50 values for these extracts (12.50 ± 0.36, 13.65 ± 0.29, and 14.27 ± 0.21 µg/mL, respectively) were comparable with that of the positive control-Na 2 EDTA*2H 2 O (IC 50 = 8.75 ± 0.15 µg/mL). Shin and coauthors also evaluated the metal chelating capacity of different parts of W. japonica. They found that the highest chelating activity was expressed by a boiled water extract of the leaves (76.9%), followed by fruits (68.9%), flowers (62.9%), and floral stalks (38.5%) [24].
The radical scavenging activities of the phenylpropanoid derivatives isolated from the leaves of W. japonica were also evaluated against superoxide anion radicals using an electron spin resonance (ESR) method. The results of this study showed that 1-

Anti-Collagenase Activity
Collagen, a dominant constituent of normal human dermis that is mainly responsible for its structural stability. Its reduction is started by collagenases, which split interstitial collagens [32]. The inhibition of this enzyme activity can decrease the collagen degradation and, thus, delay wrinkle formation in aging skin. The anti-collagenase effect of the extracts from the flowers, roots, and leaves of E. japonicum was measured using C. histolyticum collagenase. The results are presented in Table 4. Table 4. Anti-collagenase, anti-elastase, and anti-hyaluronidase activities of the flowers (WJA), roots (WJB), and leaves (annual-WJC and biennial-WJD) of E. japonicum. * Statistically significant differences compared to epigallocatechin gallate (EGCG), p < 0.05. All the investigated extracts of E. japonicum inhibited more than 70% of the collagenase activity. The extract from the flowers (93.34% ± 0.77%) and from the biennial leaves (90.16% ± 0.51%) possessed a significantly higher activity compared to the positive control-EGCG (88.49% ± 0.45%).

Sample
Numerous studies have been demonstrated that flavonoids and phenolic acids are mostly the compounds responsible for the inhibition of collagenase [33,34].

Anti-Elastase Activity
In normal adult skin, the elastin dominates, representing over 90% of the total content of the developed elastic fiber. This protein is primarily responsible for the elasticity of skin [32]. Since there are many reports showing that skin aging is directly related to the breakdown of elastin by the enzyme elastase [33], the elastase inhibitory activity was also determined for the E. japonicum extracts.
The analysis was achieved using a N-Succinyl-Ala-Ala-Ala-p-nitroanilide (substrate molecule) and elastase obtained from a porcine pancreas (enzyme). EGCG was used as a positive control, and it inhibited 91.03% ± 0.18% of the enzyme activity. All the extracts showed similar properties, but the highest activity was observed for the extract from the roots-WJB (90.18% ± 0.54%). All the results of the anti-elastase activity are presented in Table 4.

Anti-Hyaluronidase Activity
Hyaluronic acid, which is located at the periphery of the collagen and elastin fibers in young skin, disappears in aged skin. The decreases in the hyaluronic acid level, caused by an increase in the hyaluronidase activity, can result in changes in the aged skin, such as a reduced turgidity, wrinkling, and altered elasticity. Thus, hyaluronidase inhibitors are useful ingredients, as they have antiaging effects on the skin [35,36].
To the best of our knowledge, previous studies have not reported the in vitro anticollagenase, anti-elastase, and anti-hyaluronidase activities of E. japonicum. Although the influence of isosaponarin isolated from the leaves of W. japonica on collagen synthesis in human fibroblasts was investigated. The authors found that this flavone glycoside increased the type I collagen production at the mRNA gene level [20].

Antibacterial Activity
All the samples (extracts and standards) were preliminary tested for their antibacterial activity by the modified disc diffusion method, determining the zones of bacterial growth inhibition. The size of a bacterial growth inhibition zone is directly proportional to the degree of sensitivity of the bacteria against the tested drug. The larger the inhibition zone, the higher the sensitivity of the bacteria against the analyzed agent.
The data indicated that only two studied extracts had strong inhibition activity against Gram-positive, Gram-negative, and aerobic or microaerobic bacterial strains. Among the four E. japonicum extracts, only WJB and WJA possessed antibacterial activity (Table 5). This activity was especially focused on the microaerobic strains: S. mutans, S. sanguinis, P. acnes PCM 2400, and P. acnes PCM 2334, with the zones of growth inhibition ranging from 16 to 21 mm. Such a narrow spectrum of antimicrobial activity makes it possible to target the therapeutic use of the WJA and WJB extracts against anaerobic pathogens. In turn, two other extracts (WJC and WJD) showed low or no activity against the tested bacterial strains. In turn, the phenolic acids and flavonoid standards used in our experiment (quercetin-K1; luteolin-K2; gallic acid-K3; p-coumaric acid-K4; caffeic acid-K5; vanillic acid-K6), except for K3, had no activity against the tested bacterial strains. K3 showed a broad spectrum of activity against all the strains, ranging from 10 mm to 36 mm.
Moreover, the MIC (minimum inhibitory concentration) of the samples was determined for active extracts, which exhibited the bacterial growth inhibition zones. In the MIC test (Table 6), the activity of the E. japonicum extracts was determined in the range of 250-500 µg/mL against the anaerobic strains. The remaining sample determinations against the combination of microorganisms showed no significant activity. Additionally, to distinguish the inhibiting and killing abilities of the tested extracts, the MBC (minimal bactericidal concentration) was determined.
The MIC is the lowest concentration of drug that inhibits the bacterial growth, observed as no turbidity in the culture media. Additionally, the MBC is the lowest concentration that kills the bacteria. It is well-known that bacteriostatic antimicrobial agents have MBC/MIC ratios greater than or equal to 16, while bactericidal antimicrobial agents possess MBC/MIC ratios less than or equal to 4 [37]. In this study, the MBC/MIC ratios for the tested strains (Table 6) indicated that the studied extracts (WJA, WJC, and K3) exhibited a bacteriostatic mode of action against the tested pathogens.
No previous studies have been reported about the antibacterial activity of E. japonicum against the bacterial strains tested in this research. However, in a previous study, an antimicrobial protein (WjAMP-1), purified from W. japonica leaves, showed an inhibition of growth of Escherichia coli (IC 50 = 8 µg/mL) [38]. Moreover, the roots, stems, and leaves of W. japonica collected in Korea and Japan showed bactericidal activities against H. pylori strain NCTC 11637 (reference strain), YS 27 (from a duodenal ulcer patient), and YS 50 (from stomach cancer) [39].

Cytotoxic Activity
The human skin fibroblast viability after a 24-h incubation with the tested extracts is presented in Figure 1. Taking into account the obtained results, it was observed that the BJ cells showed different responses to the extracts in concentration-dependent manner. In general, most of investigated extracts were nontoxic, with CC 50 values above 1000 µg/mL (Table 7). Nevertheless, in the case of the WJB extract, it was found that it slightly inhibited the BJ cell viability (Figure 1), with a CC 50 value close to 254 µg/mL (Table 7). Thus, it was proven that the tested extracts (apart from WJB) were nontoxic towards normal human fibroblasts, and as a consequence, they can be considered very promising substances for further investigations.
In the previous research, the cytotoxic activities of nine compounds isolated from the roots of W. japonica, collected in the Republic of Korea, were evaluated against A549 (non-small-cell lung adenocarcinoma), SKOV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and BT549 (invasive ductal carcinoma) cell lines using the sulforhodamine B (SRB) bioassay. In this research, the authors found that trans-p-coumaric acid possessed a cytotoxic activity against the BT549 cell line, with an IC 50 value equal to 10 µM. The other tested compounds (wasabiside A-E, trans-ferulic acid, benzoic acid, and syringic acid) were inactive (IC 50 > 10 µM) against all the tested cancer cell lines used [27]. (Table 7). Nevertheless, in the case of the WJB extract, it was found that it slightly inhibited the BJ cell viability (Figure 1), with a CC50 value close to 254 μg/mL (Table 7). Thus, it was proven that the tested extracts (apart from WJB) were nontoxic towards normal human fibroblasts, and as a consequence, they can be considered very promising substances for further investigations.

Plant Material
The flowers, leaves (annual and biennial), and roots of Eutrema japonicum (Miq.) Koidz. were collected from the Wasabi Farm Poland in Jedlnia Letnisko (Poland) at an altitude of 160.3 m a.m.s.l., coordinates 51 • 43 52" N, 21 • 32 27" E, in January 2021. Taxonomical identification was confirmed by Prof. K. Szewczyk, the botanist from the Department of Pharmaceutical Botany (Medical University in Lublin, Poland). Voucher specimen was deposited in the Department of Pharmaceutical Botany (EJ-250121).

Preparation of the Extracts
The plant materials were dried in the shade at 24 • C (±0.5 • C) to achieve a constant weight [40]. Extracts were prepared using a mixture of methanol-acetone-water (3:1:1, v/v/v; 3 × 100 mL) and were then sonicated at a controlled temperature (40 ± 2 • C) for 30 min [25]. The combined extracts were filtered, concentrated under reduced pressure, and, after freezing, lyophilized in a vacuum concentrator (Free Zone 1 apparatus; Labconco, Kansas City, MO, USA) to obtain dried residues. Dry extracts were weighted and stored in a freezer at −20 • C. The following yields were obtained: flowers (WJA)-1.17 g, roots (WJB)-3.41 g, annual leaves (WJC)-2.41 g, and biennial leaves (WJD)-4.71 g.

Total Flavonoid and Phenolic and Phenolic Acid Contents
Total flavonoid (TFC) and total phenolic contents (TPC) were established using the colorimetric assays, as described previously [25]. The absorbances were measured at 430 and 680 nm, respectively, using a Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). TPC was estimated from the calibration curve (R2 = 0.9845) using gallic acid as a standard (concentration range 0.002-0.1 mg/mL). The results were expressed as the mg of gallic acid equivalents (GAE) per 1 g of dry extract (DE). TFC was estimated from the calibrated curve (R2 = 0.995) using quercetin (0.004-0.11 mg/mL) as the standard. The results were expressed as the mg of quercetin equivalents (QE) per 1 g of DE. The total phenolic acids (TPAC) content was assessed using Arnov's reagent, as described in Polish Pharmacopoeia IX (an official translation of PhEur 7.0) [40]. The absorbance was measured at 490 nm. The TPAC was estimated from the calibration curve (R2 = 0.9999) using caffeic acid as a standard in the concentration of 3.36-23.52 µg/mL. The results were expressed as the mg of caffeic acid equivalents (CAE) per 1 g of DE.

LC-ESI-MS/MS Analysis
The Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA, USA) coupled to a 3200 QTRAP mass spectrometer (AB Sciex, Redwood City, CA, USA) was used for the qualitative and quantitative analyses of flavonoids and phenolic acids in different parts of the E. japonicum extracts. The separation of the analyzed compounds, injected in a 3-µL amount, was performed on a Zorbax SB-C18 analytical column (2.1 × 100 mm, 1.8 µm; Agilent Technologies, Palo Alto, CA, USA) at 25 • C. Elution was carried out using solvent A (0.1% HCOOH in water) and solvent B (0.1% HCOOH in acetonitrile). The following gradient elution program was used: 0-2 min-20% B, 3 to 4 min-25% B, 5 to 6 min-35% B, 5 to 6 min-35% B, 8-12 min-65% B, 14-16 min-80% B, and 20-28 min-20% B. The flow rate was 300 µL/min. The mass spectra of the analyzed compounds were acquired in the negative ESI mode, and the optimum values of the source parameters were as follows: capillary temperature 450 • C, nebulizer gas 50 psi, curtain gas 30 psi, and source voltage −4500 V for phenolic acids and flavonoid glycosides and capillary temperature 550 • C, nebulizer gas 30 psi, curtain gas 20 psi, and source voltage −4500 V for the flavonoid aglycones analysis. Details of the LC-ESI-MS/MS analysis are presented in Tables 8 and 9. Analyst 1.5 software (AB Sciex, Redwood City, CA, USA) was used for the analysis and data acquisition. Table 8. LC-ESI-MS/MS analytical results of the phenolic acids and flavonoids.

Antioxidant Activity
All antioxidant and enzyme inhibitory assays were done in 96-well plates (Nunclon, Nunc, Roskilde, Denmark) using an Infinite Pro 200F Elisa Reader (Tecan Group Ltd., Männedorf, Switzerland). The experiments were performed in triplicate.

DPPH• Assay
The 2,2-diphenyl-1-picryl-hydrazyl (DPPH•) free radical scavenging activity of the E. japonicum extracts and positive control-ascorbic acid (AA)-was studied using the method described previously [25] but with some modifications. After 30 min of incubation at 28 • C, the decrease in DPPH• absorbance, caused by the tested extracts, was measured at 517 nm. The results were expressed as the values of the IC 50 .

ABTS •+ Assay
The ABTS •+ decolorization assay was the second method applied for an assessment of the antioxidant activity [25]. The absorbance was measured at 734 nm. Trolox was used as a positive control. The results were expressed as the values of the IC 50 .

Metal Chelating Activity (CHEL)
The metal chelating activity was established using the method described by Guo et al. [41], modified in our previous study [25,42]. The absorbance was measured at 562 nm. As a positive control, Na 2 EDTA*2H 2 O was used. The results were expressed as the IC 50 values of the E. japonicum extracts based on the concentration-inhibition curves.

Anti-Hyaluronidase Activity
Anti-hyaluronidase activity was established using the method described by Liyanaarachchi et al. [45]. After 20 min of incubation at 37 • C, the absorbance was measured at 585 nm. Epigallocatechin gallate (100 µg/mL) was used as a positive control.

Bacterial Strains
All the tested extracts were screened for their in vitro antibacterial activity against strains: aerobic Gram-positive Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, aerobic Gram-negative Escherichia coli ATCC 25992, Pseudomonas aeruginosa ATCC 27853, microaerobic Gram-positive Propionibacterium acnes PCM 2400, Propionibacterium acnes PCM 2334, S. mutans PCM 2502, and S. sanguinis PCM 2335. For determination of the antibacterial activity, Mueller-Hinton agar or broth (MH-agar or MH-broth) for aerobic and Brain-Heart Infusion agar or broth (BHI-agar or BHI-broth) for microaerobic strains were used. Bacterial inoculum was prepared by subculturing microorganisms into MH-agar or BHI-agar at 37 • C for 24 h or 48 h, respectively. The growth was harvested using 5 mL of 0.9% NaCl and diluted to 0.5 McFarland (1.5 × 10 8 CFU/mL (CFU: colony-forming unit)).

Agar Disc Diffusion Assay
The antibacterial character of all the samples was initially determined by a disc diffusion assay [46]. The bacterial inoculum was spread on the agar surface of the Petri plates by applying a cotton swab. The solutions of all the tested samples (100 µg/mL in DMSO) were placed on inoculated Petri plates. The plates with MH-agar (for aerobic strains) were incubated for 24 h at 37 • C, while the plates with BHI-agar were incubated for 48 h at 37 • C. After incubation, the diameter of the growth inhibition zone around the samples was measured.

MIC (Minimum Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) Analysis
The minimum inhibitory concentration (MIC) of the extracts was determined for the bacterial strains, which exhibited the bacterial growth inhibition zones. The test was performed using double-serial microdilution in the 96-well microtiter plates, according to the CLSI method but with some modifications [47]. Two hundred microliters of broth were pipetted into each well. The double-serial dilution of the tested derivatives was performed in the test wells, resulting in concentrations that ranged from 2000 µg/mL to 0.098 µg/mL. Finally, 2 µL of tested bacteria inoculum were added to the wells (except for the negative sterility control and blanc dye control). The tests were performed at 37 • C for 24 h (aerobic strains) or for 48 h (microaerobic strains). After incubation, the panel was digitally analyzed at 600 nm using the microplate reader BioTek™ Synergy™ H4 Hybrid Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The growth intensity in the sample well was compared to the negative and positive controls. Additionally, the MBC (minimal bactericidal concentration) was determined by spreading 10 µL of liquid from a clear sample well on the agar medium. The plates were incubated for 24 h at 37 • C, and the MBC was defined as the lowest concentration of sample without bacterial growth. Each experiment was repeated in triplicate.

Cytotoxicity Evaluation
The cytotoxic effect of the tested extracts was assessed in accordance with our procedure described earlier [48,49]. The experiment was carried out using the BJ cell line (normal human skin fibroblasts, ATCC CRL-2522 TM ). Briefly, the BJ cells were seeded on a 96-well plate at a concentration of 1.5 × 10 4 cells/well and incubated for 24 h at 37 • C in a humidified atmosphere (5% CO 2 , 95% air). On the next day, stock solutions of extracts (100 mg/mL) in dimethyl sulfoxide (DMSO) were obtained, and then, two-fold serial dilutions of extracts (1000-1.95 µg/mL) in the culture medium were prepared. Subsequently, the culture medium was replaced by an appropriate dilution of extracts, and the treated BJ cells were incubated for 24 h at 37 • C, 5% CO 2 , 95% air. Cytotoxicity of the tested extracts was evaluated using the MTT assay [50]. Based on the obtained results, the values of the half-maximum cytotoxic concentration (CC 50 ) were determined (4-parameter nonlinear regression analyses, GraphPad Prism 5, version 5.04, Software, GraphPad Software, San Diego, CA, USA). The CC 50 refers to a concentration of extract required for the reduction of BJ cell viability to 50%.

Statistical Analysis
The results were expressed as the mean values ± standard deviations (SD) of three independent experiments. The data from the cell culture experiments were subjected to a statistical analysis using an unpaired Student's t-test, and the differences were considered significant when p < 0.05 (GraphPad Prism 5, version 5.04 Software, GraphPad Software, San Diego, CA, USA).

Conclusions
In this in vitro study, we characterized the composition and comprehensively evaluated the biological activities of the extracts from the flowers (WJA), roots (WJB), and leaves-annual (WJC), as well as biennial (WJD)-of Eutrema japonicum cultivated at Wasabi Farm Poland. Thus, the content of flavonoids and phenolic acids in the extracts was determined, as well as the antioxidant, anti-collagenase, anti-elastase, anti-hyaluronidase, antimicrobial, and cytotoxic properties of such extracts.
Among the tested samples, the WJA extract possessed the most desirable biological activities. It exhibited the greatest antioxidant features, as proven by the DPPH • assay, ABTS •+ test, and Metal Chelating Activity test. Moreover, it had the greatest anti-collagenase and anti-hyaluronidase features compared to the other investigated extracts. The determined MIC and MBC values proved that the WJA extract also exhibited the most promising antibacterial activity, as it possessed bacteriostatic activity against the anaerobic pathogens. In turn, the cell culture experiments indicated that the WJA extract was noncytotoxic towards normal skin human fibroblasts. The beneficial biological properties of the WJA extract seemed to be correlated with its composition. We proved that WJA is rich in flavonoids, especially isovitexin 4'-O-glucoside, luteolin 3 ,7 -diglucoside, and kaempferol 3-O-rutinoside. It is worth underlining that the aforementioned properties revealed in this study allowed us to suppose that the WJA extract has "multidirectional" activity. Thus, considering a broad spectrum of activities of such an extract, it seems to be a promising antiaging agent. For this reason, the WJA extract will be subjected to further in vivo studying in order to confirm its beneficial activity.