Anticancer and Anti-Neuroinflammatory Constituents Isolated from the Roots of Wasabia japonica

Wasabi (Wasabia japonica (Miq.) Matsum.) is a pungent spice commonly consumed with sushi and sashimi. From the roots of this plant, a new 2-butenolide derivative (1) and 17 previously reported compounds (2–18) were isolated and structurally characterized. Their chemical structures were characterized based on the conventional NMR (1H and 13C, COSY, HSQC, and HMBC) and HRESIMS data analysis. All of these phytochemicals (1–18) were evaluated for their antiproliferative effects on the four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and MKN-1), for their inhibitory activity on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 microglia cells, and for their nerve growth factor (NGF)-releasing effect from C6 glioma cells. Among the isolated compounds, compound 15 showed powerful antiproliferative activities against A549 and SK-MEL-2 cell lines with IC50 values of 2.10 and 9.08 μM, respectively. Moreover, the new compound 1 exhibited moderate NO inhibition activity with IC50 value of 45.3 μM.


Introduction
Wasabia japonica (Miq.) Matsum., commonly known as wasabi, is one of the most well-known species among Brassicaceae plants. This plant is a perennial plant and has been cultivated mainly in Korea and Japan. Because of their tangy taste, the roots of wasabi have long been used as a spice for sushi and sashimi. A wide range of biological studies on W. japonica have been investigated so far; however, most of the experiments were focused on its sulfur-containing constituents, isothiocyanates (ITCs). For examples, two major components, 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and allyl isothiocyanate (AITC), exhibited anticancer [1][2][3][4][5], antioxidant [6], anti-inflammatory [7,8], neuroprotective [9], and antimicrobial [10] properties. The other minor constituents of W. japonica contain antioxidant phenylpropanoid [11], anticancer and anti-inflammatory monogalactosyl diacylglycerides [12], and antifungal indole derivatives [13]. However, minor bioactive components of W. japonica with different structural classes other than ITCs remain largely unknown.
In our continuing efforts to discover bioactive constituents from the Korean traditional medicinal plants, we have investigated the roots of W. japonica and isolated structurally unique thioglycosides and lignan glycosides with neurotrophic and/or anti-inflammatory Antioxidants 2022, 11, 482 2 of 11 activities [14,15]. To discover anticancer compounds within non-ITCs class from this plant, we have tested antiproliferative effects of hexanes-, chloroform (CHCl 3 )-, ethyl acetate (EtOAc)-, and n-butanol (n-BuOH)-soluble fractions of the methanol (MeOH) extract of W. japonica roots, and among them, the hexanes-soluble faction displayed the most potent activities with GI 50 values of 30.57, 21.71, 16.34, and 50.64 µg/mL against A549, SK-OV-3, SK-MEL-2, and HCT-15 cancer cell lines, respectively (Table 1). Therefore, we then focused mainly on the hexanes-soluble fraction, and herein we report the isolation and structure characterization of anticancer and anti-inflammatory constituents from the W. japonica roots. A total 18 compounds were identified (Figure 1), and the structure of the new isolate, wasabolide (1), was elucidated by the conventional spectroscopic (i.e., NMR) and spectrometric (i.e., MS) data analysis.
Antioxidants 2022, 11, x FOR PEER REVIEW 2 of 11 turally unique thioglycosides and lignan glycosides with neurotrophic and/or anti-inflammatory activities [14,15]. To discover anticancer compounds within non-ITCs class from this plant, we have tested antiproliferative effects of hexanes-, chloroform (CHCl3)-, ethyl acetate (EtOAc)-, and n-butanol (n-BuOH)-soluble fractions of the methanol (MeOH) extract of W. japonica roots, and among them, the hexanes-soluble faction displayed the most potent activities with GI50 values of 30.57, 21.71, 16.34, and 50.64 μg/mL against A549, SK-OV-3, SK-MEL-2, and HCT-15 cancer cell lines, respectively (Table 1). Therefore, we then focused mainly on the hexanes-soluble fraction, and herein we report the isolation and structure characterization of anticancer and anti-inflammatory constituents from the W. japonica roots. A total 18 compounds were identified (Figure 1), and the structure of the new isolate, wasabolide (1), was elucidated by the conventional spectroscopic (i.e., NMR) and spectrometric (i.e., MS) data analysis.

Plant Material
The roots of W. japonica (3.3 kg) were collected in Hanam, Republic of Korea, in October 2014. One of the authors, Kang Ro Lee, identified the plant, and a voucher specimen (SKKU-NPL 1409) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Republic of Korea.
The results are averages of three independent experiments, and the data are expressed as mean ± SD. 3 N Gmonomethyl-L-arginine (L-NMMA) was used as a positive control.

Nerve Growth Factor (NGF) Assay
The nerve growth factor (NGF) assay was performed analogously, as described in Reference [21]. C6 glioma cell lines were used to measure the NGF of the culture medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) in 5% CO 2 incubator. The cells were seeded in a 24-well culture plate (1 × 10 5 cells/well) and incubated for 24 h. The cells were treated with or without 20 µM of the compounds (1-18), together with serum-free Dulbecco's modified Eagle's medium (DMEM) for another 24 h. Released NGF levels from the supernatants from each cell were measured by using an ELISA development kit (R&D System, Minneapolis, MN, USA). Moreover, the cell viability was evaluated by using MTT assay; 6-shogaol used as a positive control, and the results are expressed as percentage of the control group.

Structure Elucidation of Compounds 1-18
Wasabolide (1) was obtained as a colorless gum, and its molecular formula was established as C 14 Figure  S1). The 1 H NMR spectrum of 1 ( Figure S2 Figure 2A). The HMBC correlation between OCH 3 -4 and C-4 indicated a methoxy group located at the γ-position of UBL moiety (Figure 2A). In addition, an unusually smaller coupling constant between the cisoriented two olefinic protons H-2 and H-3, 5.7 Hz, was observed. The similar J value has been reported from other UBL-containing organic molecules [16,22,23], supporting the presence of UBL functionality in 1. The remaining COSY correlations between H-6 and H-5/H-7 and H-10 and H-9/H-11 and HMBC cross-peaks between H-5 and C-6, H-11 and C-9/C-10/C-12, and OCH 3 -12 and C-12 indicated the presence of a fully saturated aliphatic chain with a terminal methyl ester group as the other substructure of 1. Therefore, the planar structure of 1 was determined as in Figure 1. The most structurally similar molecule to 1 was found to be 4-methoxy-2-eicosen-4-olide (MEO) isolated from a marine sponge ( Figure 2B, right) [16] by the literature search, using SciFinder. MEO shared most of the NMR features, with 1 especially at the UBL functionality (Table 2 and Figure 2B), thus supporting our structural assignment of 1. Finally, it was concluded that 1 was a racemic mixture based on the almost zero value of specific rotation and no Cotton effect observation in electronic circular dichroism (ECD) spectrum of 1.

Biosynthetic Proposal of the New Compound 1
Upon the structural characterization of the new metabolite 1, its biosynthetic pathway was proposed as described below (Figure 3). First, one unit of acetyl-CoA and five units of malonyl-CoA could form the C12-fatty acid i with a double bond and a carbonyl group at α, βand γ-position, respectively, which reaction resembled typical fatty acid biosynthesis. Then, oxidation of the terminal methyl group in i to carboxylic acid could afford dicarboxylic acid ii [40,41]. Geometrical isomerization could occur in ii that transforms the Eto Z-configuration of the double bond to yield iii. A hemi-acetalization reaction on iii could produce iv [22,42], which contains UBL moiety, and, finally, 1 could be formed by O-methylation at both hydroxy and carboxylic groups in iv.

Anti-Neuroinflammatory Activity of the Isolated Compounds (1-18)
The potential anti-neuroinflammatory effects of the isolates (1−18) were tested by measuring the level of NO production in LPS-stimulated BV-2 microglia cells. As shown in Table 4, among the tested phytochemicals, the new compound 1 showed the strongest inhibitory activity on the NO production, with an IC50 value of 45.3 μM and without significant cell toxicity up to 20 μM. Compounds 5, 11, and 15 also exhibited weak activity with IC50 value of 90.0, 59.6, and 92.4 μM, respectively. The other compounds were inactive (IC50 > 100 μM).

Antiproliferative Activity of the Isolated Compounds (1-18)
In line with the potent antiproliferative activity of the hexanes-soluble fraction mentioned above, the isolated compounds (1-18) were evaluated for their antiproliferative activity against four human tumor cell lines, namely A549 (non-small cell lung adenocarcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and MKN-1 (adenosquamous carcinoma), by SRB assay. Compounds 10, 11, 14-16, and 18 isolated from the hexanes-soluble fraction strongly inhibited proliferation in cancer cell lines with different selectivity (Table 3). In detail, α-tocospiro A (15) showed potent activity against A549 cell line with IC 50 value of 2.10 µM, which was comparable to that of the positive control substance, etoposide (1.51 µM). While the chemical structures of 13-15 are similar in that these three molecules had the same C15 fully saturated farnesyl unit, we observed no and weaker activity of 13 (IC 50 > 20 µM) and 14 (IC 50 = 13.28 µM), respectively, on the same cancer cell line. These data suggested that the bulky spirobicyclic moiety in α-tocospiro A (15) would play an important role in exhibiting antiproliferative activities in A549 cell line. Interestingly, Yuan et al. reported that α-tocospiro A (15) had no inhibitory activity on the A549 proliferation (IC 50 > 20 µM) [43], but we observed the significant potency of α-tocospiro A (15) in this study. Moreover, α-tocospiro A (15) also displayed a strong antiproliferative effect on SK-OV-2, SK-MEL-2, and MKN-1 cell lines, with IC 50 values ranging from 9.08 to 13.23 µM . Compounds 10, 11, 14, 16, and 18 showed antiproliferative activities on several cancer cell lines with IC 50 values between 12.86 to 26.72 µM. The other compounds were inactive (IC 50 > 30 µM).

Conclusions
From the roots of W. japonica, a total 18 compounds (1-18), including a new phytochemical (1), were isolated by chromatographic methods and identified by spectroscopic and spectrometric data analysis, including 1D and 2D NMR and HRMS. The structure of the new compound 1 was assigned as a possibly dicarboxylic acid-derived 2-butenolide derivative and its biosynthetic pathway was also proposed based on the assigned structure and related literature search. Compound 1 showed strong anti-neuroinflammatory activity by inhibiting NO production in LPS-stimulated BV-2 cells and α-tocospiro A (15) exhibited a potent antiproliferative activity against A549 non-small cell lung adenocarcinoma cells.