Current Topics: Regular Article
Identification of Acidic Triterpenoid Saponins from Silene vulgaris Using a Methylation-Based Isolation and LC-MS Analysis Strategy
2025 Volume 73 Issue 3 Pages 179-188
Details
2025 Volume 73 Issue 3 Pages 179-188
The Silene genus plants in the Caryophyllaceae family are a large genus with over 850 species. It has been known that Silene genus plants contain triterpenoid saponins. These saponins have glucuronic acid in the sugar chain and are difficult to separate in chromatography. In this study, a strategy was developed to clarify the distribution of triterpenoid saponins in whole plants of Silene vulgaris by isolation of neutralized saponins using methylation reactions, which were used as standard substances for LC-MS analysis, and elucidating their characteristic MS and MS/MS fragment patterns. The n-butanol fraction of the methanol extract from the whole plant of S. vulgaris was separated to obtain fractions including saponins by octadecyl silica column chromatography. Then, each fraction was treated with trimethylsilyl diazomethane for methylation of the carboxyl groups of glucuronic acid in the molecules, and five triterpenoid methylated saponins (7a, 13a, 14a, 16a, and 17a) were isolated using HPLC. The chemical structures of the isolated compounds were determined by spectroscopic analyses including NMR and MS, and their characteristic fragmentations were also clarified in LC-MS and MS/MS. It was performed on the n-butanol fraction from the whole plant of S. vulgaris, and the chemical structures of 22 triterpenoid saponins were estimated based on the MS and MS/MS fragmentation patterns of the isolated triterpenoid saponins.
The genus Silene (Caryophyllaceae) has long been known as a rich source of triterpenoid saponins, with historical records indicating their use as detergents due to their surfactant properties.1) Despite the genus comprising approximately 850 species,2) detailed phytochemical investigations into the triterpenoid saponins of Silene species remain limited. To date, around 60 triterpenoid saponins have been isolated and structurally characterized from 9 Silene species, including S. armeria,3) S. coeli-rosa,4) S. fortune,5,6) S. gallica,7) S. jenisseensis,8,9) S. rubicunda,10–15) S. szechuensis,16) and S. viscidula.17,18)
During our ongoing phytochemical studies on saponins from the Caryophyllaceae family, we have successfully isolated and structurally elucidated eight triterpenoid saponins, including four novel compounds from S. rubicunda,13) and twelve triterpenoid saponins, including seven novel compounds from S. armeria.3) However, throughout these studies, we encountered significant challenges in the isolation of acidic saponins, which contain glucuronic acid as part of their sugar moiety. These saponins exhibited poor chromatographic separation, even under acidic conditions, complicating their purification and structural elucidation.
Recent advances in LC-MS technologies, particularly high-resolution (HR) mass spectrometry, have greatly facilitated the structural analysis of natural products.19–22) While LC-MS has been instrumental in the rapid identification of plant metabolites, it has limitations when it comes to determining the precise sugar composition and sequence of saponins, especially in the absence of reference standard compounds.23)
To address these challenges, a strategy was developed for the identification of acidic triterpenoid saponins in Silene species, with S. vulgaris selected as a model plant for applying this approach. First, carboxyl groups in the acidic saponin fractions were methylated to yield neutral saponin derivatives. These derivatives were then separated by column chromatography and subjected to structural elucidation through spectroscopic analyses, such as NMR. Subsequently, the methylated saponins were used as reference standards to establish their chromatographic behavior and characteristic ion fragmentation patterns in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Finally, based on these findings, the extract was analyzed by LC-MS/MS to enable the rapid and accurate identification of acidic triterpenoid saponins.
Silene vulgaris Garcke (Synonyms: S. cucubalus Wibel, S. inflata Sm., S. venosa Asch.), a perennial herb native to Europe, was introduced to Japan as an ornamental plant during the early Meiji period and has since become naturalized.24) The plant grows to a height of 60–90 cm and bears white, tubular flowers that bloom from May to July. While primarily cultivated as an ornamental plant, its young shoots and leaves are also consumed as vegetables in various European countries, including Turkey, Italy, Austria, Germany, and Spain.25) In addition, S. vulgaris has been used in folk medicine for the treatment of anemia.26) Phytochemical studies of S. vulgaris have identified seven triterpenoid saponins, all of which are acidic and contain glucuronic acid in their sugar moieties.27–29)
In the present study, the methylation-based isolation and LC-MS analysis strategy was applied to investigate the acidic triterpenoid saponins of S. vulgaris. This approach led to the isolation of five methylated triterpenoid saponins (7a, 13a, 14a, 16a, and 17a) and, through LC-MS/MS analysis, the identification of 22 triterpenoid saponins (1–22), including 18 previously unreported compounds (1–7, 9–11, 13, 15, 16, 18–22) (Fig. 1).
The whole plants of S. vulgaris were extracted with methanol (MeOH), and the resulting extract was subjected to liquid–liquid partitioning to yield ethyl acetate (EtOAc)-, n-BuOH-, and water-soluble fractions. The n-BuOH-soluble fraction was further fractionated using C18 column chromatography (CC). Fractions containing acidic triterpenoid saponins were treated with trimethylsilyldiazomethane (TMSCHN2) and subsequently separated by reverse-phase HPLC, affording five methylated triterpenoid saponins: 7a, 13a, 14a, 16a, and 17a. Their structures were elucidated through extensive spectroscopic methods, including HR-electrospray ionization (ESI)-MS, and 1D and two-dimensional (2D)-NMR experiments.
Compound 7a was identified as a previously reported compound, quillaic acid 3-O-α-l-arabinopyranosyl-(1→3)-O-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid dimethyl ester.30) Since only partial NMR data for 7a are available in the literature, fully assigned 1H- and 13C-NMR data are shown in Tables 1–4.
| Position | 7a | 13a | 14a | 16a | 17a |
|---|---|---|---|---|---|
| 1 | α 0.85 (m) | α 0.86a) | α 0.84a) | α 0.83a) | α 0.83a) |
| β 1.39a) | β 1.39 (m) | β 1.39a) | β 1.41a) | β 1.42a) | |
| 2 | α 2.01(m) | α 2.04 (m) | α 2.01 (m) | α 2.03 (m) | α 2.04 (m) |
| β 1.79 (m) | β 1.80 (m) | β 1.78a) | β 1.80 (m) | β 1.83 (m) | |
| 3 | 4.01 (dd, 12.5, 4.9) | 4.04 (dd, 10.9, 3.5) | 4.06a) | 4.02 (dd, 11.9, 4.5) | 4.04 (dd, 12.9, 6.3) |
| 5 | 1.33a) | 1.39a) | 1.38a) | 1.35a) | 1.36a) |
| 6 | α 0.90 (m) | α 0.99a) | α 0.98a) | α 0.95 (m) | α 0.94 (m) |
| β 1.33a) | β 1.39a) | β 1.38a) | β 1.35a) | β 1.35a) | |
| 7 | α 1.43a) | α 1.23a) | α 1.23a) | α 1.29a) | α 1.29 (m) |
| β 1.10 (br d, 12.6) | β 1.07a) | β 1.05 | β 1.01 (br d, 12.3) | β 1.01 (br d, 12.0) | |
| 9 | 1.74 (br d, 9.2) | 1.67 (dd, 10.6, 7.2) | 1.68 (dd, 10.6, 7.2) | 1.55 (dd, 15.2, 6.1) | 1.55a) |
| 11 | 1.87a) | α 1.75 (m) | A 1.80a) | 1.87 (2H, m) | 1.86 (2H, m) |
| β 1.98 (m) | B 1.95 (m) | ||||
| 12 | 5.46a) | 5.40 (t, 3.2) | 5.40 (t, 2.6) | 5.35 (t, 3.4) | 5.34 (t, 3.5) |
| 15 | α 1.54 (dd, 14.5, 2.3) | α 2.10 (d, 15.5) | α 2.12 (d, 15.5) | α 1.93 (d, 14.6) | α 1.93 (brd, 14.3) |
| β 1.92a) | β 2.60 (d, 15.5) | β 2.62 (d, 15.5) | β 2.51a) | β 2.50a) | |
| 16 | 4.95 (br s) | ||||
| 17 | 1.89a) | 1.92a) | 2.51a) | 2.50a) | |
| 18 | 3.32 (dd, 13.8, 3.2) | 2.34a) | 2.37 (m) | 2.80 (m) | 2.80 (ddd, 14.0, 6.3, 4.0) |
| 19 | α 1.25a) | α 0.96 (m) | A 0.96a) | α 1.44a) | α 1.45a) |
| β 2.69 (t, 13.8) | β 1.89 (m) | B 1.89a) | β 1.19, m | β 1.17a) | |
| 21 | α 2.38 (td, 13.2, 4.9) | α 1.39a) | A 1.10 (m) | α 1.61 (dd, 13.5, 4.0) | α 1.61 (dd, 13.5,4.0) |
| β 1.25a) | β 1.12a) | B 1.39a) | β 1.12 (m) | β 1.12 (m) | |
| 22 | α 2.17 (br d, 12.6) | α 2.34a) | α 2.30 (m) | α 2.16 (m) | α 2.15 (br d, 13.5) |
| β 1.89a) | β 1.39a) | β 1.37a) | β 1.35a) | β 1.35a) | |
| 23 | 9.85 (s) | 9.95 (s) | 9.96 (s) | 9.93 (s) | 9.93 (s) |
| 24 | 1.39 (s) | 1.46 (s) | 1.46 (s) | 1.45 (s) | 1.45 (s) |
| 25 | 0.80 (s) | 0.80 (s) | 0.80 (s) | 0.826 (s)b) | 0.831 (s)b) |
| 26 | 0.77 (s) | 0.73 (s) | 0.73 (s) | 0.865 (s)c) | 0.869 (s)c) |
| 27 | 1.68 (s) | 1.02 (s) | 1.01 (s) | 1.15 (s) | 1.16 (s) |
| 29 | 0.97 (s) | 0.95 (s) | 0.94 (s) | 0.832 (s)b) | 0.835 (s)b) |
| 30 | 1.05 (s) | 0.85 (s) | 0.85 (s) | 0.869 (s)c) | 0.871 (s)c) |
| 28-OCH3 | 3.65 (s) |
a) Overlapped with other signals. b, c) Interchangeable.
| Position | 7a | 13a | 14a | 16a | 17a |
|---|---|---|---|---|---|
| 1 | 38.0 | 37.6 | 37.6 | 37.8 | 37.9 |
| 2 | 25.0 | 25.1 | 25.5 | 25.1 | 25.2 |
| 3 | 84.4 | 84.6 | 84.6 | 84.5 | 84.5 |
| 4 | 54.9 | 54.9 | 54.9 | 54.9 | 54.9 |
| 5 | 48.5 | 48.8 | 48.8 | 48.5 | 48.6 |
| 6 | 20.3 | 20.5 | 20.5 | 20.3 | 20.3 |
| 7 | 32.5 | 32.5 | 32.0 | 32.3 | 32.3 |
| 8 | 39.9 | 39.0 | 39.0 | 39.9 | 39.9 |
| 9 | 46.8 | 47.3 | 47.3 | 47.0 | 47.0 |
| 10 | 36.1 | 36.4 | 36.4 | 36.2 | 36.2 |
| 11 | 23.6 | 23.4 | 23.9 | 23.5 | 23.5 |
| 12 | 122.2 | 117.6 | 117.6 | 122.7 | 122.7 |
| 13 | 144.5 | 142.7 | 142.7 | 142.7 | 142.7 |
| 14 | 41.8 | 42.9 | 42.9 | 47.7 | 47.7 |
| 15 | 35.8 | 44.1 | 44.1 | 47.0 | 46.9 |
| 16 | 74.1 | 213.7 | 213.7 | 213.2 | 213.1 |
| 17 | 48.9 | 49.6 | 49.6 | 46.9 | 46.8 |
| 18 | 41.2 | 37.0 | 37.0 | 44.8 | 44.8 |
| 19 | 46.8 | 42.9 | 42.9 | 46.8 | 46.7 |
| 20 | 30.7 | 30.6 | 30.6 | 31.0 | 31.1 |
| 21 | 35.8 | 38.6 | 38.6 | 34.7 | 34.7 |
| 22 | 32.4 | 23.9 | 23.9 | 21.2 | 21.3 |
| 23 | 209.9 | 210.1 | 210.1 | 210.1 | 210.0 |
| 24 | 10.9 | 10.9 | 10.9 | 11.1 | 11.1 |
| 25 | 15.6 | 15.6 | 15.6 | 15.5 | 15.5 |
| 26 | 17.0 | 16.7 | 16.7 | 17.6 | 17.6 |
| 27 | 27.0 | 25.4 | 25.1 | 27.0 | 27.0 |
| 28 | 177.7 | ||||
| 29 | 33.1 | 33.3 | 33.3 | 33.4 | 33.4 |
| 30 | 24.5 | 24.7 | 24.7 | 23.5 | 23.5 |
| 28-OCH3 | 51.7 |
| Position | 7a | 13a | 14a | 16a | 17a |
|---|---|---|---|---|---|
| GlcA | GlcA | GlcA | GlcA | GlcA | |
| 1 | 4.83 (d, 7.2) | 4.86 (d, 7.5) | 4.87 (d, 7.5) | 4.84 (d, 7.2) | 4.85 (d, 7.5) |
| 2 | 4.22 (m) | 4.25a) | 4.29a) | 4.26a) | 4.29a) |
| 3 | 4.20a) | 4.21a) | 4.21a) | 4.19a) | 4.21a) |
| 4 | 4.20a) | 4.23a) | 4.27a) | 4.24a) | 4.27a) |
| 5 | 4.36a) | 4.39a) | 4.40 (d, 9.5) | 4.37 (br d, 9.2) | 4.38 (br d, 9.5) |
| 6-OCH3 | 3.72 (s) | 3.74 (s) | 3.74 (s) | 3.73 (s) | 3.72 (s) |
| Gal | Gal | Gal | Gal | Gal | |
| 1 | 5.45 (d, 7.8) | 5.49 (d, 7.7) | 5.52 (d, 7.7) | 5.48 (d, 7.8) | 5.51 (d, 7.8) |
| 2 | 4.39a) | 4.44a) | 4.45a) | 4.42a) | 4.44a) |
| 3 | 4.09 (dd, 9.7, 3.4) | 4.11 (dd, 9.8, 3.5) | 4.14 (dd, 9.6, 3.3) | 4.10 (dd, 9.8, 3.2) | 4.14 (dd, 9.8, 3.2) |
| 4 | 4.50 (br d, 3.4) | 4.53 (d, 3.2) | 4.55 (dd, 6.3, 3.2) | 4.53 (br d, 2.2) | 4.54 (br d, 3.2) |
| 5 | 3.94 (br t, 6.3) | 3.97 (br t, 6.0) | 4.02 (m) | 3.96 (br t, 6.3) | 4.01 (br t, 6.3) |
| 6 | A 4.37a) | A 4.39a) | A 4.44a) | A 4.41a) | A 4.44a) |
| B 4.43a) | B 4.48 (m) | B 4.49a) | B 4.48 (m) | B 4.50 (m) | |
| Ara | Ara | Xyl | Ara | Xyl | |
| 1 | 5.20a) | 5.19 (d, 7.5) | 5.27 (d, 7.7) | 5.18 (d, 7.7) | 5.26 (d, 8.3) |
| 2 | 4.40a) | 4.44a) | 3.93 (m) | 4.42a) | 3.93 (t, 8.3) |
| 3 | 4.06 (dd, 9.5, 3.4) | 4.07 (dd, 9.2, 3.5) | 4.09a) | 4.06 (dd, 9.2, 3.2) | 4.07 (t, 8.3) |
| 4 | 4.19a) | 4.21a) | 4.10a) | 4.20a) | 4.10a) |
| 5 | A 3.68 (dd, 12.9, 1.7) | A 3.71 (m) | A 3.62 (br d, 8.6) | A 3.70 (br d, 11.2) | A 3.62 (m) |
| B 4.19a) | B 4.21a) | B 4.21a) | B 4.21a) | B 4.21a) |
a) Overlapped with other signals.
| Position | 7a | 13a | 14a | 16a | 17a |
|---|---|---|---|---|---|
| GlcA | GlcA | GlcA | GlcA | GlcA | |
| 1 | 103.6 | 103.9 | 103.9 | 103.9 | 103.9 |
| 2 | 78.3 | 78.5 | 78.5 | 78.5 | 78.5 |
| 3 | 85.5 | 85.7 | 85.7 | 85.7 | 85.7 |
| 4 | 70.9 | 70.9 | 71.0 | 71.0 | 71.0 |
| 5 | 76.3 | 76.4 | 76.4 | 76.4 | 76.4 |
| 6 | 169.8 | 169.9 | 169.9 | 169.9 | 169.9 |
| 6-OCH3 | 52.1 | 52.2 | 52.2 | 52.2 | 52.1 |
| Gal | Gal | Gal | Gal | Gal | |
| 1 | 104.0 | 104.2 | 104.3 | 104.2 | 104.3 |
| 2 | 73.5 | 73.6 | 73.6 | 73.6 | 73.7 |
| 3 | 75.3 | 75.4 | 75.5 | 75.5 | 75.5 |
| 4 | 70.1 | 70.1 | 70.1 | 70.1 | 70.2 |
| 5 | 76.6 | 76.7 | 76.8 | 76.7 | 76.8 |
| 6 | 61.6 | 61.7 | 61.8 | 61.8 | 61.8 |
| Ara | Ara | Xyl | Ara | Xyl | |
| 1 | 104.8 | 105.0 | 104.9 | 105.0 | 105.0 |
| 2 | 72.7 | 72.8 | 75.2 | 72.9 | 75.3 |
| 3 | 74.5 | 74.6 | 78.5 | 74.7 | 78.6 |
| 4 | 69.4 | 69.6 | 70.8 | 69.6 | 70.8 |
| 5 | 67.5 | 67.6 | 67.3 | 67.7 | 67.3 |
Compounds 14a and 17a, previously unreported, were identified as the methyl esters of two known saponins, neogypsoside A (14)31) and its C-17 isomer, neogypsoside B (17),31) respectively. This conclusion was based on the nearly identical NMR data between the methylated derivates and the known saponins (14a vs. 14, and 17a vs. 17), with the primary differences attributed to the characteristic upfield shift of the carboxyl carbon resonance in β-glucuronic acid (GlcA) moiety due to methyl esterification (–1.2 ppm). Further confirmation was obtained through the heteronuclear multiple bond connectivity (HMBC) correlations of GlcA-6-OCH3 (14a: δH 3.74; 17a: δH 3.72) with GlcA-C-6′ (14a and 17a: δC 169.9) (Fig. 2). Thus, 14a was determined as neogypsogenin A 3-O-(β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid) methyl ester, and 17a as neogypsogenin B 3-O-(β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid) methyl ester.
Compounds 13a and 16a were also previously unreported. The structural relationship between 13a and 16a mirrors that of 14a and 17a, where the aglycones of 13a and 16a are C-17 isomers, while their sugar chains are identical. The 1H- and 13C-NMR resonances assignable to the aglycone portions of 13a and 16a closely match those of 14a and 17a, respectively, indicating that the aglycone in 13a is neogypsogenin A, and in 16a, neogypsogenin B. The relative configuration of H-17 was confirmed by key rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) correlations, with H-17/H3-27 observed in 13a indicating the α-orientation, and H-17/H3-26 in 16a indicating the β-orientation (Figs. 3, 4). In contrast, the 1H- and 13C-NMR resonances assignable to the sugar chains in 13a and 16a show high similarity to those of 7a, suggesting identical glycosyl structures. This conclusion was further supported by detailed 2D-NMR data analysis. Accordingly, 13a was determined as neogypsogenin A 3-O-(β-d-arabinopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid) methyl ester, and 16a as neogypsogenin B 3-O-(β-d-arabinopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid) methyl ester.
Compound 7a was a methylated derivate of the oleanane-type triterpenoid saponin 7, while compounds 13a, 14a, 16a, and 17a were methylated derivates of the noroleanane-type triterpenoid saponins 13, 14, 16, and 17, respectively. Notably, the peaks corresponding to saponins 7, 13, 14, 16, and 17 in the fractions disappeared after methylation treatment in LC-MS analysis. Among these, saponins 14 and 17 were previously known compounds, neogypsosides A and B, respectively,31) while saponins 7, 13, and 16 have not been reported before.
Chromatographic Behavior and MS and MS/MS Fragmentation of Methylated Triterpenoid SaponinsThe chromatographic behavior, as well as MS and MS/MS fragmentation patterns, was analyzed using isolated methylated triterpenoid saponins as reference compounds. Noroleanane-type triterpenoid saponins 13a, 14a, 16a, and 17a were identified as isomers, differing in the configuration of the aglycone at C-17 (13a vs. 16a, and 14a vs. 17a) and the composition of the sugar moiety (13a vs. 14a, and 16a vs. 17a) (Fig. 1). Comparison of the retention time (13a: 11.99 min, 14a: 12.29 min, 16a: 12.45 min, and 17a: 12.78 min) in LC-MS suggested that the saponins with an H-17α-orientation eluted earlier than those with an H-17β-orientation, while saponins containing an arabinosyl moiety eluted earlier than those containing a xylosyl moiety.
When comparing the fragmentation patterns in the MS and MS/MS, several characteristics were observed (Supplementary Table S1). In the positive ion mode of full MS, saponins (13a and 16a) containing an arabinosyl moiety showed comparable product ion intensities between the peaks corresponding to the removal of terminal Gal (m/z 763) and Ara (m/z 793). However, for those (14a and 17a) containing a xylosyl moiety, the product ion intensity from the removal of Xyl (m/z 793) was much higher than that from the removal of Gal (m/z 763). In the MS/MS analysis using aglycone-derived product ions as the precursor ions, common fragmentation patterns were observed regardless of the C-17 configuration. The fragmentation pathways included product ions resulting from dehydration reactions, as well as a series of product ions derived from the retro Diels–Alder (RDA) cleavage of the C-ring (Fig. 5A).
The MS of the oleanane-type triterpenoid saponin 7a showed a fragmentation pattern similar to those of compounds 13a and 16a, corresponding to the sugar chain. In the MS/MS, the product ion spectrum obtained from the [Aglycone + H]+ ion as the precursor ion revealed characteristic product ions derived from the A/B ring and the D/E ring via the RDA reaction. Notably, among the product ions derived from the D/E ring, a product ion resulting from the detachment of a methylated carboxy group was detected (Fig. 5B).
Analysis of Triterpenoid Saponins in the Whole Plants of Silene vulgaris Using LC-MS/MSAn LC-MS analysis of the n-BuOH fraction from the whole plants of Silene vulgaris was conducted (Figs. 6A, 6B, Supplementary Table S2). A total of 22 kinds of triterpenoid saponins were identified based on chromatographic behavior and the fragment patterns of methylated triterpenoid saponins.
Compounds 15, 18, 19, 20, 21, and 22 were suggested to have the same aglycone as 13 and 14, or 16 and 17 from MS/MS analysis using the [Aglycone + H]+ ion as a precursor ion in positive ion mode. Compounds 15 and 18, isomers of each other, are thought to have a sugar chain composed of galactose and glucuronic acid, based on their molecular formula and fragment ions derived from the sequential loss of sugar residues in full MS. The aglycone of 15 was considered to be neogypsogenin A, and those of 18 was considered to be neogypsogenin B from the relationship between retention times (15: 10.12 min vs. 18: 10.55 min). Therefore, 15 was presumed to be neogypsogenin A 3-O-β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, and 18 was to be neogypsogenin B 3-O-β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid. The molecular formulas of compounds 19–22 suggested their sugar chains containing glucuronic acid, and arabinose or xylose moieties. Retention time comparisons revealed that compounds 19 and 20 are neogypsogenin A glycosides, while compounds 21 and 22 are neogypsogenin B glycosides (19: 12.32 min and 20: 12.42 min vs. 21: 12.87 min and 22: 12.95 min). Furthermore, it was suggested that compounds 19 and 21 likely possess arabinose moieties, whereas compounds 20 and 22 likely possess xylose moieties (tR 19 vs. 20, and 21 vs. 22). Therefore, 19 was presumed to neogypsogenin A 3-O-α-l-arabinopyranosyl-(1→3)-β-d-glucopyranosiduronic acid, 20 was to neogypsogenin A 3-O-β-d-xylopyranosyl-(1→3)-β-d-glucopyranosiduronic acid, 21 was presumed to neogypsogenin B 3-O-α-l-arabinopyranosyl-(1→3)-β-d-glucopyranosiduronic acid, and 22 was to neogypsogenin B 3-O-β-d-xylopyranosyl-(1→3)-β-d-glucopyranosiduronic acid.
Compounds 1–6 and 9–11 were suggested to be saponins with neogypsogenin A or neogypsogenin B possessing an additional hydroxy group on the D/E rings as aglycones, based on the observation of product ions derived from the loss of H2O from the D/E rings in MS/MS analysis using [Aglycone + H]+ ions as precursor ions in the positive ion mode (Fig. 7). The hydroxyl group at C-29 was determined based on the structure of the artifact, 4′,5′-dihydro-16-oxo-2′H-24,28-bisnorolean-12-eno[3,4-b]furan-4′,29-diol, generated from the triterpenoid saponin by TMSCHN2 treatment, which was isolated in this study (Supplementary Fig. S41, Supplementary Table S3). Compounds 1 and 4 exhibited fragment patterns similar to those of compound 13, compounds 2 and 5 to compound 14, compounds 3 and 6 to compound 15, and compounds 9, 10, and 11 to compounds 19 and 20, respectively, about the sugar chain structures of the corresponding compounds. Based on retention times, compounds 1, 2, 3, 9, and 10 were considered to have the aglycone of 29-hydroxyneogypsogenin A possessing H-17α, whereas compounds 4, 5, 6, and 11 were thought to have the corresponding 29-hydroxyneogypsogenin B possessing H-17β (tR1: 3.18 min and 2: 3.26 min vs. 4: 4.83 min and 5: 4.97 min; 3: 3.36 min vs. 6: 5.03 min; and 9: 6.77 min and 10: 6.89 min vs. 11: 7.27 min). For compounds 1 and 2 as well as 4 and 5, their retention times further supported their respective sugar chain structures (tR1 vs. 2 and 4 vs. 5). Thus, 1 was considered to 29-hydroxyneogypsogenin A 3-O-α-l-arabinopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, 2 was to 29-hydroxyneogypsogenin A 3-O-β-d-xylopyranosyl-(1→3)-β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, 3 was to 29-hydroxyneogypsogenin A 3-O-β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, 4 was to 29-hydroxyneogypsogenin B 3-O-α-l-arabinopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]- β-d-glucopyranosiduronic acid, and 5 was to 29-hydroxyneogypsogenin B 3-O-β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, and 6 was to 29-hydroxyneogypsogenin B 3-O-β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid. On the contrary, compound 9 was considered to have an arabinose moiety in its sugar chain, while compound 10 contains a xylose moiety (tR 9 vs. 10). Although the corresponding saponin for compound 11 was not detected, compound 11 is predicted to be a triterpenoid saponin with a sugar chain consisting of glucuronic acid and either arabinose or xylose moieties, or an overlapping peak of saponins containing these sugar chains. Therefore, the chemical structure of 9 was presumed to 29-hydroxyneogypsogenin A 3-O-α-l-arabinopyranosyl-(1→3)]-β-d-glucopyranosiduronic acid, 10 was 29-hydroxyneogypsogenin A 3-O-β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosiduronic acid, and 11 was 29-hydroxyneogypsogenin B 3-O-α-l-arabinopyranosyl-(1→3)]-β-d-glucopy-ranosiduronic acid or 29-hydroxyneogypsogenin B 3-O-β-d-xylopyranosyl-(1→3)]-β-d-glucopyranosiduronic acid.
Compound 8 has the same molecular formula as 7, but based on the fragment ions derived from the sequential loss of sugar units in full MS and their retention time (7: 5.05 min vs. 8: 5.11 min), it was considered to be a saponin with quillaic acid as the aglycone, identical to that of compound 7, in which the arabinose moiety the sugar chain of compound 7 is replaced by a xylose moiety. Thus, the chemical structure of 8 was estimated to be quillaic acid 3-O-β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid. This compound has been reported as a known component of Silene cucubalus.29) Compound 12 is a saponin possessing the aglycone of gypsogenin, which has one less hydroxyl group than quillaic acid, based on the molecular formula and fragment patterns of MS and MS/MS using an aglycone-derived fragment ion, and the chemical structure of 12 was presumed to be the known compound gypsogenin 3-O-β-d-xylopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic acid, which has been reported to be a component of Silene genus plants.27,29)
These compounds have an aldehyde group of C-23. Among them, the oleanane-type triterpenoid saponins have a carboxyl group of C-28, and some have a hydroxyl group at the C-16 position. On the contrary, the noroleanane type has a carbonyl group of the C-16 and is thought to have been produced biosynthetically by decarboxylation of the carboxy group of C-28. The sugar chains of saponins in this study have a disaccharide or trisaccharide consisting of glucuronic acid and galactose and/or arabinose or xylose moieties.
In this study, a methylation-based isolation and LC-MS analysis strategy was applied to investigate acidic triterpenoid saponins in Silene vulgaris. As a result, a total of 22 kinds of oleanane-type or noroleanane-type saponins were identified, including 18 previously unreported compounds. This study not only provided a rapid and effective method for the identification of acidic saponins in Silene plants but also expanded our understanding of their distribution within this genus.
Trimethylsilyldiazomethane (TMSCHN2, approx. 10% in hexane, approx. 0.6 mol/L) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 0.1 vol% formic acid-acetonitrile and 0.1 vol% formic acid-distilled water for LC-MS were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Octadecyl silica (ODS) (Chromatorex DM1020T) was purchased from Fuji Silysia Chemical Ltd. (Aichi, Japan). Pyridine-d6 was purchased from Cambridge Isotope Laboratories, Inc. MeOH, EtOAc, n-BuOH, acetonitrile (MeCN), and trifluoroacetic acid (TFA) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
General Experimental ProceduresThe optical rotations were measured using a JASCO P-2200 polarimeter (Jasco, Tokyo, Japan). UV spectra were recorded using a JASCO V-730BIO spectrophotometer. IR spectra were recorded using a JASCO FT/IR-4100 Fourier transform infrared spectrometer with the KBr disk method. 1H- and 13C-NMR spectra were recorded using a JEOL ECA-500 spectrometer (1H at 500 MHz; 13C at 125 MHz). 1H- and 13C-NMR chemical shifts are in ppm. HPLC was performed using an C18 column [Pegasil ODS SP100 column (Senshu, Inc., Tokyo, Japan)(15 cm ×20 mm i.d.) with MeCN/H2O (55 : 45) with 0.06% TFA, a flow rate of 6.0 mL min–1 (system I), and Capcell pak C18 column (Siseido, Inc., Tokyo, Japan) (15 cm ×20 mm i.d.) with MeCN/H2O (50 : 50) with 0.06% TFA (system II) and MeOH/H2O (80 : 20) with 0.06% TFA (system III), a flow rate of 5.0 mL min–1].
Plant MaterialsThe whole plants of Silene vulgaris were produced at the Toho University Medicinal Plant Garden, Chiba, Japan, were collected in May 2014, and identified by one of the authors, WL. A voucher specimen (TH-SVWP-1) was deposited at the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Toho University, Japan.
Extraction and IsolationThe whole plants of S. vulgaris (dry weight: 0.74 kg) were ultrasonically extracted with MeOH (1 h, 3 times) at room temperature, and then evaporated in vacuo. The combined extract (31.6 g) was then partitioned four times between EtOAc (500 mL) and H2O (500 mL), followed by partitioned four times between n-BuOH (500 mL) and H2O (500 mL). The n-BuOH soluble fraction (6.5 g), obtained after concentration of the solvent, was purified by CC [ODS (60 g); MeOH/H2O (4 : 6, 5 : 5, 6 : 4, 7 : 3, 8 : 2, 9 : 1, and 1 : 0); and EtOAc/MeOH (10 : 1 and 0 : 1) in a decreasing polarity order] resulting in 12 fractions (Fr. B1–B12).
B3 (212.6 mg), which were eluted using MeOH/H2O (5 : 5, respectively), dissolved in MeOH (8.5 mL), and reacted with TMSCHN2 (4.5 mL) at room temperature for 1h. Preparative HPLC (System I) of BM3 (221.8 mg), obtained after the concentration of the solvent, gave 7a (5.0 mg).
B8 (104.9 mg), which was eluted using MeOH/H2O (7 : 3 and 8 : 2), dissolved in MeOH (2 mL) and reacted with TMSCHN2 (1 mL) at room temperature for 1h. Preparative HPLC (System II) of BM8 (82.3 mg), obtained after the concentration of the solvent, gave BM8-1–BM8-6. Among them, BM8-3 (5.4 mg, tR 42.0 min), BM8-4 (2.7 mg, tR 46.4 min), and BM8-6 (2.7 mg, tR 55.6 min) were determined as 13a, 14a, and 17a. BM8-5 (7.3 mg, tR 50.4 min) was subjected to re-preparative HPLC (System III) and gave 16a (1.6 mg, tR 95.0 min).
Quillaic Acid 3-O-(α-l-Arabinopyranosyl-(1→3)-[β-d-galactopyranosyl-(1→2)]-β-d-glucopyranosiduronic Acid) Dimethyl Ester (7a)Amorphous solid;
Amorphous solid;
Amorphous solid;
Amorphous solid;
Amorphous solid;
UHPLC was conducted using a Vanquish UHPLC system (Thermo Scientific, Waltham, MA, U.S.A.). Chromatographic peaks were separated on a TSKgel ODS-120H (100 × 2.0 mm I.D., 1.9 μm) at a flow rate of 0.4 mL/min at 40°C with a column temperature oven. A mobile phase consisted of eluent A (distilled water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid) programmed as follows: 0–20 min, 30→70% B. The injection volume was 2 μL for analysis of the n-BuOH soluble fraction at 1 mg/mL of concentration.
A Q-Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometer system (Thermo Scientific, Waltham, MA, U.S.A.) with an ESI source was operated in the positive and negative ion modes. The calibration solutions were used to calibrate the ESI-MS to increase mass accuracy. The optimized parameters of mass spectrometry were illustrated below: spray voltage, +3.5 kV (for positive ion mode) or –2.5 kV (for negative ion mode); capillary temperature, 262.5°C; sheath gas flow rate, 50 units; AUX gas flow rate, 12.5 units; sweep gas flow rate, 2.63 units; S-lens RF level, 50 units; and probe heater temperature, 425 °C. Data were collected in the Full MS modes and Full MS/data-dependent (dd) MS/MS. In-source CID was set at 0 eV. The resolution was 70000 for Full MS and 35000 for Full MS/dd-MS/MS. The AGC was set at 1E6 for Full MS and 1E5 for dd-MS/MS. Maximum IT was set at 200 ms for Full MS. Scan range was set at 150 to 2000 m/z for Full MS. Data-dependent scan was performed using high energy collision dissociation (HCD) with NCE at 15 and 25 eV.
This research was supported by the Japan Society for the Promotion of Science KAKENHI 24K09867 (W. L). We thank Mr. Tsuyoshi Kawakami (Faculty of Pharmaceutical Sciences, Toho University) for his assistance with plant cultivation.
WL and KK conceptualized the study. WL designed the study. DL, TK, and KO conducted the experiments. TK and WL drafted the manuscript. All authors reviewed and approved the final manuscript.
The authors declare no conflict of interest.
This article contains supplementary materials.
