LC–MS profiling of glucosinolates in the seeds of Brassica elongata Ehrh., and of the two stenoendemic B. botteri Vis and B. cazzae Ginzb. & Teyber

Abstract The glucosinolates (GLs) present in seed extracts of Brassica elongata Ehrh., B. botteri Vis and B. cazzae Ginzb. & Teyber from Croatia were identified by LC–MS. 4-Hydroxyindol-3-ylmethyl GL (3) was the major GL in the seeds of B. elongata, along with the four minor GLs 2-(R)-hydroxy-3-butenyl- (1), 3-butenyl- (2), 4-pentenyl- (4) and indol-3-ylmethyl (5). The seeds of B. botteri (Vis island) and B. cazzae (Sušac island) contained 2 as the major GL as well as 1, 3, 5 and 4-methoxyindol-3-ylmethyl GL (6). However, the GLs in B. botteri (Palagruža island) differed from other varieties having 2-propenyl GL (7) as the major GL in the seeds, and the four minor GLs 2, 3, 5 and 6. This first report of the GL content in the seeds of B. elongata, B. botteri and B. cazzae indicates that the unique GL profiles could be specific to the geographical origin of the plant.

GLs are sulphur-containing secondary metabolites that are present in all plants of the order Brassicales, and known to be responsible for various biological activities by their degradation products (mostly ITCs) (Blažević et al. 2016). The aim of the present study was to identify the GLs in the seeds of B. elongata, B. botteri and B. cazzae growing wild on Croatian open-sea islands. The GLs were extracted and analysed as intact GLs by LC-MS.

Results and discussion
The extraction of the seeds of B. elongata, B. botteri and B. cazzae and the LC-MS analysis of intact GLs (Zrybko et al. 1997) were performed as described in the supplementary material section. The retention time (t R ), uV and mass spectra of each product were compared with those of standards from our GL library (Figures 1, S1-S3 and Table S1). Five GLs (1-5) were identified in B. elongata and six (1-3 and 5-7) in B. botteri and B. cazzae.
Compounds 1 (t R 7.6 min), 2 (t R 18.3 min) and 7 (t R 8.2 min) had identical t R , uV and mass spectra as those of commercial standards of 2-(R)-hydroxy-3-butenyl-, 3-butenyl-and 2-propenyl GLs, respectively. Compound 3 (t R 20.9 min) had a uV spectrum identical to the one of an authenticated standard of 4-methoxyindol-3-ylmethyl GL but with a different t R . In the same LC-MS conditions, the t R for 4-methoxyindol-3-ylmethyl GL stands in the region 29-30 min (Montaut et al. 2010). In addition, the mass spectrum of compound 3 gave a mass of 463 [M] − showing a difference of 14 amu with the mass of 4-methoxyindol-3-ylmethyl GL (477 [M] − ), thus indicating that compound 3 is 4-hydroxyindol-3-ylmethyl GL. Compound 4 at t R 22.9 min had identical t R , uV and mass spectra as those of 4-pentenyl GL previously identified in our group (Blažević et al. 2013). Compounds 5 (t R 25.3 min) and 6 (t R 29.2 min) had identical t R , uV and mass spectra as those of indol-3-ylmethyl GL and 4-methoxyindol-3-ylmethyl GL, respectively, both being previously isolated in our group (Montaut et al. 2010).
The results of our investigations (Table S2) show that 3 is the major GL (85%) in the seeds of B. elongata, along with the four minor GLs 1 (6.6%), 2 (3.8%), 4 (2.0%) and 5 (2.6%). In a previous report, the presence of 2-hydroxy-3-butenyl GL was mentioned in the seeds of B. elongata (Horn & Vaughan 1983); however, our study brings the precision that the GL aglycon is R-configurated i.e. 1. Furthermore, we did not detect any arylaliphatic or sulfanylalkyl GLs, which may be due to genetic and environmental factors. B. elongata grown in Croatia does not seem to produce arylaliphatic GLs which are biosynthesised from tyrosine and phenylalanine but instead produces 3 (major) and 5 (minor) derived from tryptophan along with other minor GLs (1, 2 and 4) derived from dihomomethionine (Blažević et al. 2016). Moreover, indole GLs were identified in the seeds, whereas no previous study mentioned their presence in B. elongata. This can be explained by the high instability of indol-3-ylmethyl ITCs which precludes theirs detection by GC (agerbirk et al. 2009).
our investigations (Table S2) showed that the seeds of B. botteri (Vis island) and B. cazzae (Sušac island) contained 2 as the major GL (45.0-64.3%) as well as 1, 3, 5 and 6. our results obtained with samples from Vis and Sušac islands confirmed that 2 is the major GL in the seeds as it was observed by others in B. incana (Horn & Vaughan 1983). Furthermore, we did not identify the same minor GLs. However, the major GL in the seeds of B. botteri (Palagruža island) was found to be 7 (43.5%), together with the four minor GLs 2, 3, 5 and 6. These differences may also be due to genetic and environmental factors. It seems that B. botteri and B. cazzae grown in Croatia produce major GLs (2 or 7) derived from dihomo methionine and minor GLs derived from tryptophan (3, 5 and 6). Therefore, the analysis of GLs of seeds appears to be a good tool to discriminate these different varieties of B. incana growing on the open-sea Croatian islands.

Conclusion
The LC-MS analysis of the GLs in the seeds of B. elongata, B. botteri and B. cazzae growing wild in Croatia enabled the identification of seven known GLs. This first report of the GLs in the seeds of B. elongata and two stenoendemic plants i.e. B. botteri and B. cazzae from Croatia indicates that the unique GL profiles could be specific to the geographical origin of the plant.

Supplementary material
Experimental details relating to this paper are available online, alongside Tables S1-S2 and Figures S1-S3.