Glucosinolates of Sisymbrium officinale and S. orientale

Glucosinolates (GSLs) from Sysimbrium officinale and S. orientale were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS. Eight GSLs were identified in S. officinale, including Val-derived (glucoputranjivin) and Trp-derived (4-hydroxyglucobrassicin, glucobrassicin, 4-methoxyglucobrassicin, and neoglucobrassicin) as the major ones followed by Leu-derived (Isobutyl GSL), Ile-derived (glucocochlearin) and Phe/Tyr-derived (glucosinalbin). Different S. orientale plant parts contained six GSLs, with Met-derived (progoitrin, epiprogoitrin, and gluconapin) and homoPhe-derived (gluconasturtiin) as the major ones, followed by glucosinalbin and neoglucobrassicin. GSL breakdown products obtained by hydrodistillation (HD) and microwave-assisted distillation from S. officinale, as well as isopropyl isothiocyanate, as the major volatile in both isolates, were tested for their cytotoxic activity using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Generally, all volatile isolates showed similar activity toward the three cancer cell lines. The best activity was shown by isopropyl isothiocyanate at a concentration of 100 µg/mL after 72 h of incubation, with 53.18% for MDA-MB-231, 56.61% for A549, and 60.02% for the T24 cell line.


Introduction
Glucosinolates (GSLs) are sulfur-and nitrogen-containing plant-specialized metabolites found in crucifers. Through their degradation products, mostly isothiocyanates, they are recognized as cancer prevention agents and biopesticides and are responsible for distinctive flavors. To date, 90 of 139 GSLs found in plant kingdoms have been fully characterized by modern spectroscopy techniques [1][2][3]. The structural diversity of GSLs is due to variation in chain-elongation and addition of methylene groups, and secondary modification (hydroxylation, methylation, oxidation, or desaturation in most cases) in the biosynthetic pathway of different amino acid precursors. Aliphatic GSLs represent the largest group derived mostly from methionine, while branched GSLs derived from isoleucine, leucine, or valine are more scarce. Arylaliphatic GSLs are derived from phenylalanine or tyrosine and indole GSLs from tryptophan, while some still have uncertain precursors [1]. The biosynthesis of GSLs has been well studied in Arabidopsis [4,5], Brassica rapa [6], and B. oleracea [7]. Aromatic GSL biosynthesis is less studied compared to aliphatic and indolic GSLs, which featured most prominently in the literature to date [5,8]. In addition, all knowledge gathered about GSL biosynthesis derives from homology with Arabidopsis and pertains to only a small percentage of the GSLs discovered. Therefore, data about the genes responsible for the biosynthesis of 'exotic' GSLs, such as (R)-4-(cystein-S-yl)butyl GSL (glucorucolamine), 3-methoxybenzyl GSL (glucolimnanthin), or others are completely absent from the literature [8].
The genus Sisymbrium (Brassicaceae family) comprises ca. 94 species disjunctly distributed in the Old (41 spp.) and the New World (53 spp.), among which 9 spp. are known to be wild-growing in Croatia [9,10]. The center of Sisymbrium diversity is the Irano-Turanian region [11]. The migration of Sisymbrium from the western Irano-Turanian floristic region to the Mediterranean coincides with the massive desiccation of the Mediterranean Sea, which started in the middle Miocene (13.0 million years ago) and ended with the beginning of the Pleistocene (around 5.33 million years ago). This resulted in a higher diversification occurrence of the Mediterranean Sisymbrium species. A clade consisting of Mediterranean Sisymbrium damascenum, S. orientale, S. macroloma, and S. volgense points toward a common ancestor of these species with S. officinale, which emerged much earlier (in the middle Pliocene) [11].
The aim of this study was to identify and quantify GSLs from wild-growing S. officinale and S. orientale in different plant parts by their desulfo-counterparts using UHPLC-DAD-MS/MS. Furthermore, S. officinale volatiles produced after hydrodistillation and microwaveassisted distillation were analyzed using GC-MS. The antiproliferative activity of these isolates and isopropyl isothiocyanate was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against three human cancer cell lines (lung A549, bladder T24 and breast MDA-MB-231).

Glucosinolates and Volatile Constituents
The GSLs of Sisymbrium officinale and S. orientale were qualitatively and quantitatively analyzed using UHPLC-DAD-MS/MS by their desulfo counterparts. The GSLs detected in S. officinale are given in Tables 1 and 2, and the ones found in S. orientale are given in Table 3. The structures of the corresponding GSLs are shown in Figure 1. The obtained chromatograms, as well as MS 2 spectra of the obtained desulfoGSLs, are given in Figures S1-S4.
In total, eight GSLs were detected in S. officinale. An aromatic GSL, glucosinalbin (23), and indole GSLs, namely glucobrassicin (43), 4-hydroxyglucobrassicin (28), 4-methoxyglucobrassicin (48), and N-methoxyglucobrassicin (47), were identified by direct comparison to standards. Additionally, the important fragments being Na + adduct ions used to identify and recognize all desulfoGSLs detected are annotated in more detail in the Supplementary data ( Figure S4). Usual fragmentation of the thioglucosidic bond produces thioGlc fragment m/z 219 (type b), while the cleavage on the other side leads to [anhydroGlc + Na] + at m/z 185 (type a) and type c fragment arising from loss of an anhydroGlc, [M-162 + Na] + [19]. The characteristic loss of OCH 3 radical from N-methoxy indole GSL of d47 (m/z 390), as well as the loss of formaldehyde (m/z 391) can be observed in difference to C-methoxy indole GSL (d48) ( Figure S4). Isopropyl isothiocyanate, the breakdown product of glucoputranjivin (56), was detected as the major volatile after thermal degradation in volatile samples obtained by hydrodistillation, as well as after microwaveassisted distillation, and was confirmed by direct comparison to standard as well. Isobutyl GSL (62) and sec-butyl GSL (61) were detected with MS 2 spectra of the corresponding desulfoglucosinolates, which are identical ( Figure S4) but have different retention times, t R = 5.30 min and t R = 5.64 min, respectively, which is in accordance with literature data [19]. Although limited, the identification of GSLs via the MS spectra of appropriate degradation products was very helpful in this case. Isobutyl isothiocyanate and sec-butyl isothiocyanate, having the same M + (115) can be easily distinguished, as sec-butyl isothiocyanate produces characteristic fragment m/z 86 which is absent in the case of isobutyl isothiocyanate (not detected in the obtained volatile isolates,   Glucosinolates (µmol/g DW)   Figure 1. a Compound identified by MS 2 spectra and t R comparison with standard. All chromatograms are given in Figure S1 (Split sample) and Figure S2 (Krka sample), while MS 2 spectra are given in Figure S4. tr-traces; n.d.-not detected; DW-dry weight of plant material. Data are expressed as the mean value ± standard error (n = 3). Generally, isopropyl GSL (56), originating from Val biosynthesis, was the major GSL in all plant parts of S. officinale, ranging from 1.60 to 13.54 µmol/g DW in the Split sample and from traces to 7.40 µmol/g DW in the Krka sample. Other branched GSLs, such as isobutyl GSL (62), derived from Leu biosynthesis, and sec-butyl GSL (62), derived from Ile biosynthesis, were detected as minor GSLs.
Indole GSLs were detected with glucobrassicin (43) as the major one, ranging from 0.36 to 5.72 µmol/g DW. It was previously the only indole GSL detected in this plant, as far as the authors know [12]. In this study, three additional indole-type GSLs, 4-hydroxy-indol-3ylmethyl GSL (28), 4-methoxyindol-3-ylmethyl GSL (48), and N-methoxyindol-3-ylmethyl GSL (47), were identified for the first time.  Figure 1. a Compound identified by MS 2 spectra and t R comparison with standard. All chromatograms are given in Figure S3, while the MS 2 spectra are given in Figure S4. tr-traces; n.d.-not detected; DW-dry weight of plant material. Data are expressed as the mean value ± standard error (n = 3). homo-higher homologue of specified amino acids.
GSL 43 and its derivatives are usually found in many Brassica plants but not as dominant ones. Key enzymes for the modification of 43 are cytochrome P450 monooxygenases of the CYP81F subfamily and indole glucosinolate O-methyltransferases (IGMTs) of the plant O-methyltransferase family 2. CYP81Fs carry out hydroxylation reactions either in position 4 or 1 of the indole ring of 43, and IGMTs convert hydroxy to methoxy groups [20][21][22]. CYP81F1 to CYP81F3 are capable of converting 43 to 28, which is further metabolized by O-methyltransferases (IGMT1 to IGMT4) to 48. On the other hand, CYP81F4 is capable of hydroxylating nitrogen (N) at position 1. This GSL was not detected. It is known that unsubstituted N-hydroxyindoles tend to be highly reactive, and stabilization is achieved by the conversion of the N-hydroxy group into an N-methoxy group, which is catalyzed by IGMT5 [23][24][25].
Glucosinalbin (23), the only arylaliphatic GSL, was identified in the root part of the Split sample. It can be suggested that this GSL originates directly from Tyr biosynthesis, although it can also be biosynthesized indirectly by hydroxylation of glucotropaeolin, which originates from Phe biosynthesis. However, glucotropaeolin was not identified.
In total, six GSLs were detected in S. orientale using the standards. The main GSLs in S. orientale were derived from Met biosynthesis (Table 3). According to the known biosynthesis of model plant Arabidopsis thaliana, Met is elongated into 2homoMet in the first biosynthetic step, after which 4-(methylsulfanyl)propyl GSL (glucoibervirin) is formed in the core structure biosynthesis pathway. The side chain of this GSL is then oxidized by flavin-containing monooxygenase, forming 4-(methylsulfinyl)propyl GSL (glucoiberin). These GSLs were not identified in the plant tissues. Conversion of the latter is regulated by the AOP2 genes, resulting in the identified but-3-enyl GSL (12). Hydroxylation of 12, regulated by GS-OH genes, leads to progoitrin (24R) and the main detected GSL epiprogoitrin (24S).
Sisymbrium orientale also contained two arylaliphatic GSLs. Gluconasturtiin (105), originating from homoPhe biosynthesis and GSL 23, was found only in traces in roots, which can originate from Phe or Tyr biosynthesis, as previously reported.

Antiproliferative Activity
The antiproliferative activity of the volatile isolates obtained from S. officinale by hydrodistillation (HD) and microwave-assisted distillation (MAD), as well as isopropyl isotiocyanate, their major volatile, were tested against three human cancer cell lines (lung A549, bladder T24, and breast MDA-MB-231).
The best cytotoxic effect on all three cell lines was achieved after 72 h of incubation, but there was no difference in the action of the samples obtained using different isolation methods (Figure 2). The effectiveness of samples obtained from S. officinale and isopropyl isothiocyanate did not always correspond to the increase in concentration and incubation time and, in some cases, cell recovery occurred. Isopropyl isothiocyanate had a cytotoxic effect on all cell lines at a maximum concentration of 100 µg/mL after 72 h of incubation, with 53.18% for MDA-MB-231, 56.61% for A549, and 60.02% for the T24 cell line. A previous report of Lunnaria annua HD isolate, having 92% of isopropyl ITC, did not reach IC 50 at 100 µg/mL for MDA-MB-231 and A549 cell lines tested, which is in accordance with this study [26]. Di Sotto et al. reported the antimutagenic activity of S. officinale extract, 56, and isopropyl isothiocyanate against direct-acting (methyl methanesulfonate) and indirect-acting mutagens (2-aminoanthracene and 2-aminofluorene) in various Escherichia coli strains. The latter two are considered pro-carcinogenic compounds [27]. and isopropyl isothiocyanate against direct-acting (methyl methanesulfonate) and indirect-acting mutagens (2-aminoanthracene and 2-aminofluorene) in various Escherichia coli strains. The latter two are considered pro-carcinogenic compounds [27].

Isolation of Volatiles
The volatiles from the Split sample (aerial part) were isolated by two approaches: hydrodistillation (HD) and microwave-assisted distillation (MAD). Hydrodistillation was conducted in a Clevenger-type apparatus for 2.5 h using 50 g of ground aerial part (HD). A Milestone 'ETHOS X' microwave laboratory oven (1900 W maximum) was used for microwave-assisted distillation (MAD). A typical experiment was conducted at atmospheric pressure with 100 g of fresh plant material for 35 min at 500 W. The distillation process started after 10 min. The distillate was collected in a side-tube using a pentane trap, dried over anhydrous sodium sulfate, and stored at −20 • C, until analysis [26,28].

GC-MS Analysis
The gas chromatography system used consisted of a gas chromatograph, model 8890 GC, equipped with an automatic liquid injector, model 7693A, and a tandem mass spectrometer (MS/MS), model 7000D GC/TQ (Agilent Inc., Santa Clara, CA, USA). The samples were analysed 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, and 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 Kovats retention indices (relative to C 8 -C 40 n-alkanes for HP-5MS UI column) to those from literature and/or authentic samples, as well as by comparing their mass spectra with literature from 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 the HP-5MS UI column for analyses run in duplicate.

Cell Viability Assay (MTT)
An MTT spectrophotometric assay was performed on a microplate photometer, model HiPo MPP-96 (BioSan, Riga, Latvia), as previously described [26,28]. The cells were treated with Split S. officinale volatile isolates (HD, MAD) at concentrations of 1, 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, 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 (San Diego, CA, USA), normalizing the data by three independent measurements of untreated controls. The criteria used to categorize the activity against the tested cell lines was based on IC 50 values as follows: 20 µg/mL = highly active, 21-200 µg/mL = moderately active, 201-500 µg/mL = weakly active [30].

Conclusions
Re-investigation of Sisymbrium officinale by UHPLC-DAD-MS/MS, using a library of t R and MS/MS, enabled identification of indole-type GSLs, next to the previously known branched GSLs. Met-derived GSLs were found in S. orientale as dominant, along with Phe and Trp ones. Previous phylogenetic investigation suggested that S. officinale and S. orientale share a common ancestor and that S. officinale developed much earlier. While chemical investigation suggests quite different GSL profiles, both belong to simple, ancient GSL biosynthesis, which has been suggested to lack amino acid chain elongation, tryptophanderived GSLs, and a number of traits related to GSL biosynthesis from chain-elongated methionine. Chemical investigations of this genus are still scarce. This investigation suggests GSLs' (bio)diversification through their evolution. However, in order to get a full picture, further chemical investigations should be conducted on other Sisymbrium species.