Glucosinolates in Wild-Growing Reseda spp. from Croatia

Glucosinolates (GSLs) are a unique class of thioglucosides that evolved as defense mechanisms in the 16 families of the Brassicales order and present molecular tags which can be placed in a robust phylogenetic framework through investigations into their evolution and diversity. The GSL profiles of three Resedaceae species, Reseda alba, R. lutea, and R. phyteuma, were examined qualitatively and quantitatively with respect to their desulfo-counterparts utilizing UHPLC-DAD-MS/MS. In addition, NMR analysis of isolated 2-hydroxy-2-methylpropyl desulfoGSL (d31) was performed. Three Phe-derived GSLs were found in R. lutea, including glucotropaeolin (11) (0.6–106.69 mol g−1 DW), 2-(α-L-ramnopyranosyloxy)benzyl GSL (109) (8.10–57.89 μmol g−1 DW), glucolepigramin (22) (8.66 μmol g−1 DW in flower), and Trp-derived glucobrassicin (43) (0.76–5.92 μmol g−1 DW). The Phe-derived GSLs 109 (50.79–164.37 μmol g−1 DW), gluconasturtiin (105) (1.97 μmol g−1 DW), and 11 (tr), as well as the Trp-derived GSL glucobrassicin (43) (3.13–11.26 μmol g−1 DW), were all present in R. phyteuma. R. alba also contained Phe-derived 105 (0.10–107.77 μmol g−1 DW), followed by Trp-derived 43 (0.85–3.50 μmol g−1 DW) and neoglucobrassicin (47) (0.23–2.74 μmol g−1 DW). However, regarding the GSLs in R. alba, which originated from Leu biosynthesis, 31 was the major GSL (6.48 to 52.72 μmol g−1 DW) and isobutyl GSL (62) was the minor GSL (0.13 to 1.13 μmol g−1 DW). The discovered Reseda profiles, along with new evidence provided by GSL characterizations, were studied in the context of the current knowledge on GLSs in the Resedaceae family. With the exception of R. alba, the aliphatic GSLs of which were outliers among the Resedaceae species studied, this family typically contains GSLs derived primarily from Trp and Phe biosynthesis, which modifications resulted in GSLs unique to this family, implying presence of the specific genes. responsible for this diversification.

-"Circumstantial evidence"-reasonable but not conclusive evidence of qualitative analysis; -"Present"-qualitative analysis performed using relevant analytic methods (standards, MS, and NMR); -Qualitative and quantitative analyses performed.
-"Circumstantial evidence"-reasonable but not conclusive evidence of qualitative analysis; -"Present"-qualitative analysis performed using relevant analytic methods (standards, MS, and NMR); -Qualitative and quantitative analyses performed.
Qualitative analyses of GSLs in Resedaceae published up to 2001 were reviewed by Fahey et al. [24]. Bennett et al. (2004) reported a quantitative analysis of arylaliphatic and indolic GSLs in the seeds of Reseda luteola and R. odorata, while the O-glycosylated GSL, 2-(α-L-rhamnopyranosyloxy)benzyl GSL (109) was found only in R. lutea (25.0-50.0 µ mol g −1 dry weight, DW). Outside of the genus Reseda, GSL 109 was found in the plant Ochradenus baccatus, with 7.0 µ mol g −1 DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole  [24,25] Reseda alba [24,[26][27][28]

R. media
-"Circumstantial evidence"-reasonable but not conclusive evidence of qualitative analysis; -"Present"-qualitative analysis performed using relevant analytic methods (standards, MS, and NMR); -Qualitative and quantitative analyses performed.
-"Circumstantial evidence"-reasonable but no dence of qualitative analysis; -"Present"-qualitative analysis performed us lytic methods (standards, MS, and NMR); -Qualitative and quantitative analys Qualitative analyses of GSLs in Resedaceae published up to 2001 we Fahey et al. [24]. Bennett et al. (2004) reported a quantitative analysis of a indolic GSLs in the seeds of Reseda luteola and R. odorata, while the O-glyco (α-L-rhamnopyranosyloxy)benzyl GSL (109) was found only in R. lutea (25 dry weight, DW). Outside of the genus Reseda, GSL 109 was found in the p baccatus, with 7.0 µ mol g −1 DW in the root [36]. Two arylaliphatic GSLs (40S) and epiglucobarbarin (40R), were identified in R. luteola, with conten 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconast 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25 of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was conf luteola, despite the great evolutionary distance between them. Agerbirk et tigated the same plant and determined the presence of 40R, which in the s for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the l the analysis also revealed significant levels of the apparent hydroxybu could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from b the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringii 40S was identified as the main GSL in the leaf of R. luteola and accounted the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22 [24,[26][27][28]
-"Circumstantial evidence"-reasonable but not conclus dence of qualitative analysis; -"Present"-qualitative analysis performed using releva lytic methods (standards, MS, and NMR); -Qualitative and quantitative analyses perform Qualitative analyses of GSLs in Resedaceae published up to 2001 were review Fahey et al. [24]. Bennett et al. (2004) reported a quantitative analysis of arylalipha indolic GSLs in the seeds of Reseda luteola and R. odorata, while the O-glycosylated (α-L-rhamnopyranosyloxy)benzyl GSL (109) was found only in R. lutea (25.0-50.0 µ dry weight, DW). Outside of the genus Reseda, GSL 109 was found in the plant Ochr baccatus, with 7.0 µ mol g −1 DW in the root [36]. Two arylaliphatic GSLs, glucoba (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents rangin 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the s Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (10 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The pr of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed als luteola, despite the great evolutionary distance between them.  tigated the same plant and determined the presence of 40R, which in the seeds acc for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In ad the analysis also revealed significant levels of the apparent hydroxybutyl GSL, could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal fam the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17 40S was identified as the main GSL in the leaf of R. luteola and accounted for over the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The Molecules 2023, 28, x FOR PEER REVIEW  DW in the root [36]. Two arylaliphatic GSLs (40S) and epiglucobarbarin (40R), were identified in R. luteola, with conten 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconast 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25 of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was conf luteola, despite the great evolutionary distance between them. Agerbirk et tigated the same plant and determined the presence of 40R, which in the s for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the l the analysis also revealed significant levels of the apparent hydroxybu could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringii 40S was identified as the main GSL in the leaf of R. luteola and accounted the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22 [24]

R. odorata
Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole  (4) (4) [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them. Agerbirk et al. (2021) investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them. Agerbirk et al. (2021) investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them. Agerbirk et al. (2021) investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole Molecules 2023, 28, x FOR PEER REVIEW  [24,35 Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažev DW in the root [36]. Two arylaliphatic GSLs, glucoba (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents rangin 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the se Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (10 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The pr of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed als luteola, despite the great evolutionary distance between them.  tigated the same plant and determined the presence of 40R, which in the seeds acco for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In ad the analysis also revealed significant levels of the apparent hydroxybutyl GSL, could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal fam the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17 40S was identified as the main GSL in the leaf of R. luteola and accounted for over the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,25] Reseda alba [24,[26][27][28]

R. complicata
[24] R. crystallina [24] R. lutea [14,24,25,29] R. luteola [6,24,25,27,[30][31][32][33] R. media [24,34] R. odorata [6,24,25,27,30] R. phyteuma 1 [24,30,35] R. stricta [24,30] R. suffruticosa [30] Sesamoides interrupta 2 [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [ [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [ [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [ [24,35 Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažev DW in the root [36]. Two arylaliphatic GSLs, glucoba (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents rangin 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the s Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (10 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The pr of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed als luteola, despite the great evolutionary distance between them.  tigated the same plant and determined the presence of 40R, which in the seeds acc for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In ad the analysis also revealed significant levels of the apparent hydroxybutyl GSL, could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal fam the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17 40S was identified as the main GSL in the leaf of R. luteola and accounted for over the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The   [24,35] Ochradenus baccatus [36] * No.-Numbering system is related to the GSL numbers given in a review paper by Blažević et al. DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µ mol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µ mol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them.  investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Met-derived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µ mol g −1 of fresh plant material) [22,32]. The indole -"Circumstantial evidence"-reasonable but not conclusive evidence of qualitative analysis; Molecules 2023, 28, x FOR PEER REVIEW 3 of 12  [24,25] Reseda alba [24,[26][27][28]
Qualitative analyses of GSLs in Resedaceae published up to 2001 were reviewed by Fahey et al. [24]. Bennett et al. (2004) reported a quantitative analysis of arylaliphatic and indolic GSLs in the seeds of Reseda luteola and R. odorata, while the O-glycosylated GSL, 2-(α-L-rhamnopyranosyloxy)benzyl GSL (109) was found only in R. lutea (25.0-50.0 µmol g −1 dry weight, DW). Outside of the genus Reseda, GSL 109 was found in the plant Ochradenus baccatus, with 7.0 µmol g −1 DW in the root [36]. Two arylaliphatic GSLs, glucobarbarin (40S) and epiglucobarbarin (40R), were identified in R. luteola, with contents ranging from 0.1 to 10.0 and 25.0 to 50.0 µmol g −1 DW, respectively. In the same work, in the seeds of Caylusea abyssinica, in addition to the same arylaliphatic GSLs, gluconasturtiin (105) and 40R were found to have the highest contents (10.0-25.0 µmol g −1 DW) [25]. The presence of 40S, a characteristic GSL in the genus Barbarea (Brassicaceae), was confirmed also in R. luteola, despite the great evolutionary distance between them. Agerbirk et al. (2021) investigated the same plant and determined the presence of 40R, which in the seeds accounted for 5% of the total amount of enantiomeric glucobarbarins, i.e., 1% in the leaf. In addition, the analysis also revealed significant levels of the apparent hydroxybutyl GSL, which could be either Metderived 4-hydroxybutyl GSL ( [26]) (unknown from basal families at the time) or Leu-derived 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) [17]. GSL 40S was identified as the main GSL in the leaf of R. luteola and accounted for over 90% of the total GSLs from the leaf surface (0.5 µmol g −1 of fresh plant material) [22,32]. The indole GSL glucobrassicin (43) was found in the seeds of R. odorata in the range of 10.0-25.0 µmol g −1 DW [25]. conringiin, 31) based on a previous report by Olsen and Sorensen (1979) [27]. The MS 2 spectrum ( Figure S4A) revealed characteristic fragments, the origins of which were previously explained (a, b, and c fragments), and a fragment specific to hydroxylated aliphatic dGSLs that originated from water loss (type f, m/z 316). As no standard was available, the GSL was isolated from the flower due to its high amount (52.72 µmol g −1 DW), and NMR spectra of its desulfo-counterpart were recorded (Figure 2 and Figure S5A-D). The H-1/H-2 and H-5/H-6a/H-6b spin systems' coupling constants correspond to a thioglucosyl moiety. Additionally, two distinct doublets were found between two H-8 protons ( 2 J = 15.2 Hz) at 2.83 and 2.74 ppm, respectively. Furthermore, two singlets at 1.35 and 1.33 ppm, respectively, denote two methyl groups (H-10a and H-10b). Finally, according to 13 C NMR, the same methyl groups are represented by two peaks at 28.5 and 27.9 ppm, and the precise quaternary carbon that those methyl groups are next to may be seen at 71. 1 ppm. O-Rhamnosylated 109 and indole 43 GSLs were identified in all parts of the R. phyteuma plant. The content of 109 was significantly higher than in R. lutea, up to 164.37 µ mol g −1 DW in the flower, which is why this plant species represents a good source of this GSL. Arylaliphatic 11 and 105 were identified only in the stem and at very low concentrations.
R. alba had a markedly different GSL profile compared to the other two species of this family that have been studied. The dominant desulfoglucosinolate (dGSL) signal at tR = 1.64 min and m/z 334 was assumed to be 2-hydroxy-2-methylpropyl GSL (glucoconringiin, 31) based on a previous report by Olsen and Sorensen (1979) [27]. The MS 2 spectrum ( Figure S4A) revealed characteristic fragments, the origins of which were previously explained (a, b, and c fragments), and a fragment specific to hydroxylated aliphatic dGSLs that originated from water loss (type f, m/z 316). As no standard was available, the GSL was isolated from the flower due to its high amount (52.72 µ mol g −1 DW), and NMR spectra of its desulfo-counterpart were recorded (Figures 2 and Figures S5A-D). The H-1/H-2 and H-5/H-6a/H-6b spin systems' coupling constants correspond to a thioglucosyl moiety. Additionally, two distinct doublets were found between two H-8 protons ( 2 J = 15.2 Hz) at 2.83 and 2.74 ppm, respectively. Furthermore, two singlets at 1.35 and 1.33 ppm, respectively, denote two methyl groups (H-10a and H-10b). Finally, according to 13 C NMR, the same methyl groups are represented by two peaks at 28.5 and 27.9 ppm, and the precise quaternary carbon that those methyl groups are next to may be seen at 71.1 ppm. The m/z 318 signal in MS 2 ( Figure S4A) may correspond to two isomers, desulfoglucocochlearin (d61) and isobutyl dGSL (d62). This GSL was previously identified in Sisymbrium officinale at the same retention time (tR = 5.30 min), which is consistent with this study [37]. The m/z 318 signal in MS 2 ( Figure S4A) may correspond to two isomers, desulfoglucocochlearin (d61) and isobutyl dGSL (d62). This GSL was previously identified in Sisymbrium officinale at the same retention time (t R = 5.30 min), which is consistent with this study [37].
In evolutionary terms, Brassicales were initially only able to synthesize GSLs from Phe and branched-chain amino acids, after which the indolic GSLs, which are produced from the amino acid Trp, started to appear [10]. As can be seen from Table 1 and from the results obtained in this study, the GSLs of Resedaceae are mostly biosynthesized from Phe/Tyr and Trp. The only indole-type GSLs appear to be 43 and 47. The distribution of side-chainmodified Trp-derived GSLs in basal families is poorly understood, with Salvadoraceae and Tovariaceae as exceptions, giving some reason to believe that N-methoxylation is rather ancient [6,39]. In the case of Phe GSLs, biosynthetic diversification occurred during evolution. Glucotropaeolin (11) (R. lutea and R. phyteuma) originated directly from Phe biosynthesis, which can be further hydroxylated to produce 22 (R. lutea). The GSL 109, a Phe-derived GSL additionally glycosylated in the ortho position and with a rhamnose moiety, as a tag for the Resedaceae family, was found in high amounts in R. lutea and R. phyteuma. Furthermore, another Phe-derived GSL with an arabinose moiety was recently reported in Ochradenus baccatus, implying that plants in the Resedaceae have specific genes responsible for the glycosylation of the benzylic ring in the ortho position, which needs to be investigated further. Additionally, other GSLs are biosynthesized after the elongation of