2.1. 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.
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].
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).
The flavonoid aglycones (naringenin, apigenin, kaempferol, eriodyctiol, quercetin, taxifolin, isorhamnetin, and luteolin) were mainly observed in the flower extract. However, their amounts were below the limit of quantification (LOQ). Luteolin was observed in a quantifiable amount (0.01 ± 0.00 mg/g DE) only in the extract from the annual leaves (WJC).
Among the obtained extracts, that of the flowers of E. japonicum had the highest total flavonoid glycoside content (17.15 mg/g DE). Quercetin 3-O-rutinoside and isovitexin 4’-O-glucoside were determined in all the studied parts. The isovitexin 4’-O-glucoside content was the highest in the annual leaves and flowers extracts (6.22 ± 0.04 and 6.02 ± 0.10 mg/g DE, respectively). In the flowers extract, great amounts of luteolin 3′,7′-diglucoside and kaempferol 3-O-rutinoside were also observed (4.98 ± 0.23 and 2.50 ± 0.06 mg/g DE, respectively).
According to the literature data, there have not been many reports focusing on the identification of phenolic acids and flavonoids in
E. japonicum. However, ten flavonoids (namely, isovitexin, isosaponarin, apigenin, luteolin, isoorientin, 7-
O-
trans-sinapoylisovitexin, 6″-
O-(2-
O-trans-sinapoyl-β-D-glucopyranosyl)-7-
O-trans-sinapoylisovitexin 4′-
O-β-D-glucopyranoside, 7-
O-
trans-sinapoylisovitexin 4′-
O-β-D-glucopyranoside, 7-
O-trans-sinapoylisovitexin 4′-
O-(6-
O-
trans-sinapoyl-β-D-glucopyranoside), and 6′’-
O-(2-
O-trans-sinapoyl-β-D-glucopyranosyl)-7-
O-
trans-sinapoylisovitexin) were isolated from the fresh leaves of wasabi [
9]. Using the HPLC method, Kurata et al. [
10] identified luteolin, isorhamnetin-3-glucoside, astragalin, isovitexin, isoorientin, and rutin in the flowers of
W. japonica. Moreover, from the leaves of
W. japonica cultivated in Japan, isosaponarin [
12,
20] and isoorientin were isolated [
12]. Moreover, Hosoya et al. [
11] isolated
trans-
p-hydroxycinnamic acid,
trans-ferulic acid,
trans-sinapic acid, 3,4-dimethoxy-
trans-cinnamic acid,
trans-ferulic acid methyl ester,
trans-sinapic acid methyl ester, 3,4-dihydroxy-5-methoxy-
trans-cinnamic acid, and 3,4-dihydroxy-5-methoxy-
trans-cinnamic acid methyl ester from the leaves of
W. japonica. From the roots of
W. japonica collected in the Republic of Korea,
trans-
p-coumaric acid,
trans-ferulic acid, benzoic acid, and syringic acid were also isolated [
27].
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-p-coumaric 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.
2.2. 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.
2.2.1. 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).
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].
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
2EDTA*2H
2O (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-(3″,4″-dihydroxy-5″-methoxy)-
O-
trans-cinnamoyl-2′-
O-
trans-sinapoyl gentiobiose, 1-
O-
trans-caffeoyl-2′-
O-
trans-sinapoyl gentiobiose, 1,2′-di-(3″,4″-dihydroxy-5″-methoxy)-
O-
trans-cinnamoyl gentiobiose, 1-(3″,4″-dihydroxy-5′′-methoxy)-
O-
trans-cinnamoyl-2′-
O-
trans-feruloyl gentiobiose, 3,4-dihydroxy-5-methoxy-
trans-cinnamic acid, and 3,4-dihydroxy-5-methoxy-
trans-cinnamic acid methyl ester exhibited IC
50 values close to 29.0, 85.0, 8.0, 17.0, 36.0, and 31.0 lM, respectively. It is worth underlining that these values were significantly lower compared to the result obtained for the positive control—ascorbic acid (IC
50 = 140 lM) [
11].
2.2.2. 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.
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%).
Numerous studies have been demonstrated that flavonoids and phenolic acids are mostly the compounds responsible for the inhibition of collagenase [
33,
34].
2.2.3. 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.
2.2.4. 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].
In our study, the extract from the flowers (WJA) exhibited the best hyaluronidase inhibition activity (47.32% ± 0.53%) compared to other samples. Most importantly, such activity was comparable with that of the positive standard—EGCG (62.90% ± 0.12%). In turn, the hyaluronidase inhibition activity for the extracts from the roots (WJB) and annual and biennial leaves (WJC and WJD) was 13.46% ± 0.22%, 28.95% ± 0.69%, and 25.78% ± 0.18%, respectively.
To the best of our knowledge, previous studies have not reported the in vitro anti-collagenase, 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].
2.2.5. 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].
2.2.6. 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].