Lepidium graminifolium L.: Glucosinolate Profile and Antiproliferative Potential of Volatile Isolates

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active.

These activities are usually correlated to the presence of isothiocyanates (ITCs), but under certain conditions, those can show instability [8]. De Nicola et al. reported that in hydrodistillation-mimicking conditions, most benzylic-type ITCs underwent conversion into the corresponding benzyl alcohols or benzylamines [9].
Thus, the aim of the present work was GSL quantification in different plant parts of wild-growing L. graminifolium. GSLs were identified and quantified by their desulfocounterparts using UHPLC-DAD-MS/MS. The identification of the present GSLs was also performed using GC-MS of the volatiles produced after hydrodistillation and extraction after hydrolysis by myrosinase. Furthermore, the antiproliferative activity of L. graminifolium volatiles and benzyl ITC was investigated using the MTT method against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229.

Glucosinolates and Volatile Constituents
GSLs of L. graminifolium were qualitatively and quantitatively analyzed using UHPLC-DAD-MS/MS by their desulfo-counterparts (Table 1, Figures 1, S1 and S2). The qualitative analysis of GSLs was also confirmed by their breakdown products obtained through enzymatic and/or thermal degradation. Isothiocyanates (ITCs), nitriles and other volatiles originating from GSL degradation were identified by GC-MS. (Table 2).
Five minor GSLs 1-5 biosynthetically originate from Met. 1, 3 and 4 were found only in the sample collected from Rab Island, while they were absent in Split samples. They were also present in all parts (inflorescence, stem, root, fruit) reported previously [11]. On the other hand, 2 was present only in Split samples in all parts analyzed by their desulfo-GSL and/or its breakdown product 4-(methylsulfinyl)butyl ITC (sulforaphane). Traces of 5 were found in Split root sample only (Tables 1 and 2). According to the studies of biosynthetic route in the plant model Arabidopsis thaliana, Met-derived GSLs start with eight  Table 1).   [4]. Spectra are given in Figure S4.
Five minor GSLs 1-5 biosynthetically originate from Met. 1, 3 and 4 were found only in the sample collected from Rab Island, while they were absent in Split samples. They were also present in all parts (inflorescence, stem, root, fruit) reported previously [11]. On the other hand, 2 was present only in Split samples in all parts analyzed by their desulfo-GSL and/or its breakdown product 4-(methylsulfinyl)butyl ITC (sulforaphane). Traces of 5 were found in Split root sample only (Tables 1 and 2). According to the studies of biosynthetic route in the plant model Arabidopsis thaliana, Met-derived GSLs start with eight enzymatic steps in order to elongate the chain by two C atoms, followed by core GSL biosynthesis leading to the parent dihomoMet derived GSL, 4-(methylsulfanyl)butyl GSL (5). Further sequential secondary modifications of the parent GSL, via 4-(methylsulfinyl)butyl GSL (2) and but-3-enyl GSL (4) end with 2-hydroxybut-3-enyl GSL (mixture of two stereoisomers, 1 and 3) [1,22]. In our case the L. graminifolium collected in June 2021 (when it starts growing) contained 2 and 5, while samples collected in October 2016 contained 1 and 3 with traces of 4, indicating that the time of harvest may display a snapshot of a different step of the GSL biosynthetic pathway in the plant.
Other volatiles included 4-hydroxy-3,5-dimethoxybenzaldehyde and 3-methoxybenzal dehyde, which can be formed due to the instability of the corresponding ITCs during the isolation and/or GC conditions. 4-Hydroxy-3,5-dimethoxybenzaldehyde can be converted from 4-hydroxy-3,5-dimethoxybenzyl alcohol due to the presence of the electron-donating OH group in para position (Figure 2).  [4]. Spectra are given in Figure S4.
Other volatiles included 4-hydroxy-3,5-dimethoxybenzaldehyde and 3-methoxybenzaldehyde, which can be formed due to the instability of the corresponding ITCs during the isolation and/or GC conditions. 4-Hydroxy-3,5-dimethoxybenzaldehyde can be converted from 4-hydroxy-3,5-dimethoxybenzyl alcohol due to the presence of the electrondonating OH group in para position (Figure 2).

Antiproliferative Activity
The antiproliferative activity of volatile isolates obtained from Rab Island L. graminifolium was tested against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229 (Figure 3) using MTT assay and IC50 values were calculated. According to the IC50 values, the antiproliferative activities of hydrodistillate and extract against UM-UC-3 cells after 72 h were ca. 100 μg/mL, which can be considered as moderately active. IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active. Based on the GC-MS analysis, it can be suggested that the formation of nitriles in HD (87.41%), mostly phenylacetonitrile (85.72%) from 9 (instead of benzyl ITC, 6.04%), resulted in lower antiproliferative activities. Similarly, EXT contained high percentages of nitriles (48.76%) in comparison to ITCs (5.82%), mostly from 9 and 12 which suggested the same conclusion. However, regulation of cell proliferation, cell cycle, and apoptosis plays crucial roles in the ITC-induced anti-cancer effects, and such phenomena are mainly regulated by complex mechanisms involving caspases, Bcl-2 family proteins, and mitochondrial activities [23]. Benzyl ITC has shown antiproliferative and proapoptotic activity in bladder cells [24,25]. The treatment of UM-UC-3 cells with benzyl ITC and phenylethyl ITC at low micromolar concentrations caused the damage of both outer and inner mitochondrial membranes, leading to the release of cytochrome c into the cytoplasm and caspase-9 activation as the major step leading to induction of apoptosis in this cell line [24]. Additionally, benzyl ITC mitochondrial damage is regulated by various members of

Antiproliferative Activity
The antiproliferative activity of volatile isolates obtained from Rab Island L. graminifolium was tested against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229 (Figure 3) using MTT assay and IC 50 values were calculated.
6, x FOR PEER REVIEW 6 of 11 on with Wiley/NIST library. c Compound identified by mass spectra comparison with literature values [4]. Specven in Figure S4.
Other volatiles included 4-hydroxy-3,5-dimethoxybenzaldehyde and 3-methoxybenzaldehyde, which can be formed due to the instability of the corresponding ITCs during the isolation and/or GC conditions. 4-Hydroxy-3,5-dimethoxybenzaldehyde can be converted from 4-hydroxy-3,5-dimethoxybenzyl alcohol due to the presence of the electrondonating OH group in para position (Figure 2).

Antiproliferative Activity
The antiproliferative activity of volatile isolates obtained from Rab Island L. graminifolium was tested against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229 (Figure 3) using MTT assay and IC50 values were calculated. According to the IC50 values, the antiproliferative activities of hydrodistillate and extract against UM-UC-3 cells after 72 h were ca. 100 μg/mL, which can be considered as moderately active. IC50 of benzyl ITC was 12.3 μg/mL, which can be considered as highly active. Based on the GC-MS analysis, it can be suggested that the formation of nitriles in HD (87.41%), mostly phenylacetonitrile (85.72%) from 9 (instead of benzyl ITC, 6.04%), resulted in lower antiproliferative activities. Similarly, EXT contained high percentages of nitriles (48.76%) in comparison to ITCs (5.82%), mostly from 9 and 12 which suggested the same conclusion. However, regulation of cell proliferation, cell cycle, and apoptosis plays crucial roles in the ITC-induced anti-cancer effects, and such phenomena are mainly regulated by complex mechanisms involving caspases, Bcl-2 family proteins, and mitochondrial activities [23]. Benzyl ITC has shown antiproliferative and proapoptotic activity in bladder cells [24,25]. The treatment of UM-UC-3 cells with benzyl ITC and phenylethyl ITC at low micromolar concentrations caused the damage of both outer and inner mitochondrial membranes, leading to the release of cytochrome c into the cytoplasm and caspase-9 activation as the major step leading to induction of apoptosis in this cell line According to the IC 50 values, the antiproliferative activities of hydrodistillate and extract against UM-UC-3 cells after 72 h were ca. 100 µg/mL, which can be considered as moderately active. IC 50 of benzyl ITC was 12.3 µg/mL, which can be considered as highly active. Based on the GC-MS analysis, it can be suggested that the formation of nitriles in HD (87.41%), mostly phenylacetonitrile (85.72%) from 9 (instead of benzyl ITC, 6.04%), resulted in lower antiproliferative activities. Similarly, EXT contained high percentages of nitriles (48.76%) in comparison to ITCs (5.82%), mostly from 9 and 12 which suggested the same conclusion. However, regulation of cell proliferation, cell cycle, and apoptosis plays crucial roles in the ITC-induced anti-cancer effects, and such phenomena are mainly regulated by complex mechanisms involving caspases, Bcl-2 family proteins, and mitochondrial activities [23]. Benzyl ITC has shown antiproliferative and proapoptotic activity in bladder cells [24,25]. The treatment of UM-UC-3 cells with benzyl ITC and phenylethyl ITC at low micromolar concentrations caused the damage of both outer and inner mitochondrial membranes, leading to the release of cytochrome c into the cytoplasm and caspase-9 activation as the major step leading to induction of apoptosis in this cell line [24]. Additionally, benzyl ITC mitochondrial damage is regulated by various members of the Bcl-2 family, including Bcl-2, Bax, Bak, and Bcl-xl [24]. Moreover, Tang et al. found that the urinary N-acetylcysteine conjugate of benzyl ITC suppressed different bladder cancer cells' (RT4, UM-UC-6, UM-UC-6/dox) growth through antiproliferative and proapoptotic activities. The antiproliferative mechanisms of benzyl ITC and its Nacetylcysteine conjugate were identical, but relatively longer treatment time or slightly higher doses were needed for the latter compound to exert the same effect [25].
The activity of the extracts against tested LN229 cell line observed in the same time period was similar to the one against UM-UC-3, while HD showed lower activity (Figure 3). The IC 50 of benzyl ITC was the same as against UM-UC-3 cells, i.e., 12.3 µg/mL, which can be considered as highly active. The activity of benzyl ITC against LN229 was not previously studied, as far as the authors know [26]. Zhu et al. reported that benzyl ITC can inhibit proliferation of human glioma U87MG cells, induce apoptosis and cell cycle arrest of U87MG cells, the mechanism of which may be related to the fact that benzyl ITC can cause oxidative stress to tumor cells [27]. Tang et al. reported that benzyl ITC induced cytotoxic effects through the cell cycle arrest and affected cell cycle-associated gene expression and the induction of cell apoptosis in GBM 8401 cells in vitro [28], while Shang et al. reported benzyl ITC to induce apoptosis of GBM 8401 cells via activation of caspase-8/Bid and the reactive oxygen species-dependent mitochondrial pathway [29]. Phenylethyl ITC, structural analogue of benzyl ITC, was found to inhibit the growth of LN229 cells. It was shown that this ITC can arrest the cell cycle at phase G2/M. Furthermore, it was observed that it can raise ROS expression in the tumor cells, thus suggesting that it can activate caspase-3 activity by affecting the cell cycle and inhibiting the superoxide dismutase activity as well as the glutathione expression [30].

Isolation of Desulfoglucosinolates
GSLs were extracted as previously reported [31,32]. The dried plant parts of Rab sample (aerial part) and Split sample (flower, leaf, stem, siliquae, root) were ground to a fine powder, from which 100 mg were extracted for 5 min at 80 • C in 2 × 1 mL MeOH/H 2 O (70:30 v/v) to inactivate the endogenous myrosinase. Each extract (1 mL) was loaded onto a mini-column filled with 0.5 mL of DEAE-Sephadex A-25 anion-exchange resin (GE Healthcare, Chicago, IL, USA) conditioned with 25 mM acetate buffer (pH 5.6). After washing the column with 70% MeOH and 1 mL of ultrapure water, optimal conditions for desulfation were set by adding buffer solution. Each mini-column was loaded with 20 µL (0.35 U/mL) of purified sulfatase and left to stand 18h at room temperature. The desulfo-GSLs were then eluted with 1.5 mL of ultra-pure H 2 O, lyophilized and diluted to the 1 mL. The samples were stored at -20 • C until further analysis by UHPLC-DAD-MS/MS.

UHPLC-DAD-MS/MS Analysis
Analysis was performed on UHPLC-DAD-MS/MS (Ultimate 3000RS with TSQ Quantis MS/MS detector, Thermo Fischer Scientific, Waltham, MA, USA) using Hypersil GOLD column (3.0 µm, 3.0 × 100 mm, Thermo Fischer Scientific). A gradient consisting of solvent A (50 µM NaCl in H 2 O) and solvent B (acetonitrile:H 2 O 30:70 v/v) was applied at a flow rate of 0.5 mL/min as follows: 0.14 min 96% A and 4% B; 7.84 min 14% A and 86% B; 8.96 min 14% A and 86% B; 9.52 min 5% A and 95% B; 13.16 min 5% A and 95% B; 13.44 min 96% A and 4% B; 15.68 min 96% A and 4% B. The column temperature was held at 25 • C and the injection volume was 5 µL. The electrospray interface was H-ESI source operating with a capillary voltage of 3.5 kV at 350 • C. The system was operated in the positive ion electrospray mode.

Isolation of Volatiles
The volatiles from the Rab sample (aerial part) were isolated by two approaches. Hydrodistillation was performed in Clevenger-type apparatus for 2.5 h using 50 g of dry material (HD). Dry plant material (10 g) was ground (in a coffee grinder), stirred with 20 mL of distilled water, and after endogenous and exogenous hydrolysis by myrosinase (1-2 units) for 24 h at 27 • C and pH~5.4, was extracted using CH 2 Cl 2 (EXT). pH was determined using a pH meter (Hanna Instruments, Woonsocket, RI, USA). The Split samples (flower, leaf, stem, silliquae, root) were extracted using 1 g of each plant part (EXT) [31,32].

GC-MS Analysis
The gas chromatography system used consisted of gas chromatograph, model 8890 GC, equipped with an automatic liquid injector, model 7693A, and tandem mass spectrometer (MS/MS), model 7000D GC/TQ (Agilent Inc., Santa Clara, CA, USA). The samples were analyzed on a non-polar HP-5MS UI column (dimensions: 30 m length, inner diameter 0.25 mm and stationary phase layer thickness 0.25 µm, Agilent Inc., Santa Clara, CA, USA). The column temperature program was set at 60 • C for the first 3 min and then heated to 246 • C at 3 • C/min, and maintained for 25 min isothermally. The carrier gas was helium, and the flow rate was 1 mL/min. The inlet temperature was 250 • C, while the volume of the injected sample was 1 µL. Other conditions were as follows: ionization energy was 70 eV; ion source temperature was 230 • C; the temperature of the quadrupoles was set at 150 • C. The analyses were carried out in duplicate.
The individual peaks were identified by comparison of their retention indices (relative to C 8 -C 40 n-alkanes for HP-5MS UI column) to those from a homemade library, literature and/or authentic samples, as well as by comparing their mass spectra with literature, Wiley 9N08 MS (Wiley, New York, NY, USA) and NIST17 (Gaithersburg, MD, USA) mass spectral databases. The percentages in Table 2 were calculated as the mean value of component percentages on HP-5MS UI column for analyses run in duplicate.

Cell Viability Assay (MTT)
MTT spectrophotometric assay was performed on a microplate photometer, model HiPo MPP-96 (BioSan, Riga, Latvia) as previously described [19,20]. The criteria used to categorize the activity against the tested cell lines were based on IC 50 values as follows: 20 µg/mL = highly active, 21-200 µg/mL = moderately active, 201-500 µg/mL = weakly active, and >501 µg/mL = inactive [35]. Thus, the cells were treated with Rab L. graminifolium volatile isolates (HD, EXT) at concentrations of 5, 10, 50 and 100 µg/mL in a complete medium (in triplicate) for 72 h. After treatment with isolated compounds, the cells were incubated with 0.5 g MTT/L at 37 • C for 2 h, the medium was removed, then DMSO was added, and the mixture was incubated for another 10 min at 37 • C while shaking. The degree of formazan formation, an indicator of living and metabolically active cells, was measured at 570 nm. The data were calculated in relation to the untreated control (100%) from three independent measurements. The calculation of IC 50 values was performed using GraphPad Prism software version 7.0.

Statistical Analysis
Analysis of variance (one-way ANOVA) was used to assess the statistical difference between data reported in Table 1, followed by a least significance difference test to evaluate differences between sets of mean values at significance level set at p < 0.05. Analyses were carried out using Statgraphics Centurion-Ver.16.1.11 (StatPoint Technologies, Inc., Warrenton, VA, USA) [36].

Conclusions
GSLs in L. graminifolium were quantified for the first time by UHPLC-DAD-MS/MS. In addition, new GSLs were identified in this species: glucoraphanin (2), glucoerucin (5) and one multi-substituted benzyl GSL, 4-hydroxy-3,5-dimethoxybenzyl GSL (8), which was previously reported only in L. densiflorum. Multi-substituted benzyl GSLs, such as 3,4,5-trimethoxybenzyl GSL, are rarely found but seem to be common for Lepidium species. Generally, the biosynthetic pathways are still poorly investigated, especially in most nonmodel plants, and they should be the focus of further studies. Antiproliferative effects of the tested volatile isolates rich in GSL breakdown products (mostly nitriles) on cancer cells showed moderate potential, while benzyl ITC showed high potential. Thus, different factors influencing the formation of GSL breakdown products can consequently lead to better activity of isolates containing ITCs. In addition, further experiments on benzylic-type ITCs should be performed to clarify the antiproliferative effects and underlying mechanisms.