2015 Volume 63 Issue 8 Pages 641-648
Four new resin glycosides, named calysolins XIV (1), XV (2), XVI (3), and XVII (4) were isolated from the leaves, stems, and roots of Calystegia soldanella ROEM.. et SCHULT. (Convolvulaceae). Their structures were determined based on spectroscopic and chemical evidence, and consisted of two different types: those (1) with a macrolactone structure and those (2–4) with a non-macrolactone structure. Their sugar moieties were partially acylated by specific organic acids, including tiglic, 2S-methylbutyric, and 2S,3S-nilic acids. Additionally, evaluation of the antiviral activity of 1–4 revealed effects against the herpes simplex virus type 1.
Resin glycosides are well known as purgatives, and are characteristic ingredients of crude drugs such as Pharbitis Semen, Mexican Scammoniae Radix, Orizabae Tuber, Jalapae Tuber, and Rhizoma Jalapae Braziliensis, all of which originate from Convolvulaceae plants.1) Chemical investigations on these resin glycosides were initiated as in the middle of 19th century.2) Until the middle of 1980 s, chemical studies had been confined only to characterization of the component organic acids and an oligoglycoside of a hydroxy fatty acid (glycosidic acid) afforded upon alkaline hydrolysis of mixtures of resin glycosides. In 1987, Noda et al. succeeded in the isolation and structural elucidation of four genuine resin glycosides.3) Almost all genuine resin glycosides isolated hitherto and characterized had common macrolactone structures comprising a glycosidic acid partially acylated by a few organic acids at the sugar moiety (acylated glycosidic acid). In recent years, 17 ester-type dimers of genuine resin glycosides have been isolated.4–11)
Calystegia soldanella ROEM. et SCHULT. (Convolvulaceae) is commonly found on the sandy beaches of seas and lakes located in the temperate regions of the world. The roots of this plant are used in the treatment of arthritis.12) We previously reported the isolation and structural elucidation of four new glycosidic acids including calysolic acids A–D. These compounds were obtained along with a known glycosidic acid, soldanellic acid B, and three organic acids, 2S-methylbutyric, tiglic, and 2S,3S-nilic acids, upon alkaline hydrolysis of the crude resin glycoside fraction of the leaves, stems, and roots of C. soldanella.13) We also isolated 13 genuine resin glycosides namely calysolins I–XIII, which possessed macrolactone structures, along with one known resin glycoside, soldanelline B. In addition, the antiviral activities of these compounds were evaluated against the herpes simplex virus type 1 (HSV-1).14–16) As part of an ongoing study of the resin glycosides, we further examined the constituents of this plant to clarify whether ester-type dimers are present. This report deals with the isolation and structural elucidation of four new resin glycosides named calysolins XIV (1), XV (2), XVI (3), and XVII (4). Additionally, their antiviral activity against HSV-1 is described here.
The fresh leaves, stems, and roots of C. soldanella were extracted using methanol (MeOH). This extract was suspended in H2O and then extracted successively using ethyl acetate (EtOAc) and n-butanol (BuOH). The EtOAc-soluble fraction was subjected to silica gel column chromatography as well as HPLC on octadecyl silica (ODS) and silica gel to yield 1–4.
Compound 1 was obtained as an amorphous powder. The negative-ion FAB-MS and positive-ion FAB-MS of 1 exhibited an [M−H]− ion peak at m/z 1543 and an [M+Na]+ ion peak at m/z 1567, respectively. The FAB-MS results indicated a molecular weight of 1544. The molecular formula of 1 was found to be C72H120O35, by using high-resolution (HR)-positive-ion FAB-MS. Alkaline hydrolysis of 1 yielded an organic acid fraction and a glycosidic acid. Gas chromatography (GC) analysis of the organic acid fraction revealed the presence of 2-methylbutyric, tiglic, and nilic acids. The glycosidic acid was identified as calysolic acid C (5) by comparing it with an authentic sample11) using 1H-NMR spectroscopy. The 1H-NMR spectrum of 1 exhibited signals, which indicated two H-2 [δ 2.53 (1H, ddq, J=7.0, 7.0, 7.0 Hz), 2.43 (1H, ddq, J=7.0, 7.0, 7.0 Hz)] of the 2-methylbutyryl residue, one H-2 [δ 2.77 (1H, dq, J=7.0, 7.0 Hz)] of the niloyl residue (Nla), one H-3 [δ 7.40 (1H, qq, J=1.0, 7.0 Hz)] of the tigloyl residue (Tig), and six anomeric protons [δ 6.27 (1H, d, J=1.0 Hz), 5.76 (1H, d, J=8.0 Hz), 5.08 (1H, d, J=8.0 Hz), 4.94 (1H, d, J=8.0 Hz), 4.87 (1H, d, J=8.0 Hz), 4.75 (1H, d, J=7.5 Hz)]. 13C-NMR spectrum showed signals assignable to five carboxyl carbons (δ 175.8, 175.8, 175.3, 173.2, 167.6) and six anomeric carbons (δ 105.4, 103.9, 103.5, 102.1, 101.0, 97.1). The 1H- and 13C-NMR signals were assigned on the basis of the 1H–1H correlation spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), and heteronuclear multiple bond correlation (HMBC) spectra (Tables 1, 2). A comparison of the chemical shifts of the 1H-NMR signals between 1 and the methyl ester (5a)13) of 5, revealed the downfield shifts (Δδ=δ1–δ5a) due to acylation. The signals were assignable to H-2 (Δδ=ca. 1.10) and H-4 (Δδ=1.46) of the rhamnosyl residue (Rha), H-3 (Δδ=ca. 1.34) and H-4 (Δδ=ca. 1.56) of the second glucosyl residue (Glc′), and H-2 (Δδ=1.46) of the fourth glucosyl residue (Glc‴). In addition, the 1H-NMR signals due to H2-2 of the aglycone (11S-hydroxyhexadecanoic (11S-jalapinolic) acid) moiety (Jla) of 1 were non-equivalent at δ 2.55 and 2.71, whereas 5a exhibited an equivalent signal due to H2-2 of Jla at δ 2.32 (2H, t, J=7.5 Hz).13) From these data, it was determined that 1 is composed of 1 mol each of tiglic acid, nilic acid, and 5 and 2 mol of 2-methylbutyric acid. In addition, the ester linkages were located at OH-2 and OH-4 of Rha, OH-3 and OH-4 of Glc′, and OH-2 of Glc‴. Furthermore, the carboxyl group of Jla in 1, is linked intramolecularly to a hydroxy group of the sugar moiety to form the macrolactone structure. The sites of each ester linkage of the organic acid residues and Jla were determined using the HMBC spectrum of 1. The key cross-peaks were observed between H-4 of Rha and C-1 of Tig, H-3 of Glc′ and C-1 of the first 2-methylbutyryl residue (Mba), H-4 of Glc′ and C-1 of the second 2-methylbutyryl residue (Mba′), and H-2 of Glc‴ and C-1 of Jla (Fig. 1). Although no cross-peak was observed between H-2 of Rha and C-1 of Nla in the HMBC spectrum of 1, the above data suggest that the ester linkages of Nla, Tig, Mba, Mba′, and Jla were located at OH-2 of Rha, OH-4 of Rha, OH-3 of Glc′, OH-4 of Glc′, and OH-2 of Glc‴, respectively. These linkages were confirmed by the fragment ion peaks observed at m/z 1399 [1543−144 (162 (hexosyl unit)−H2O)]−, 1069 [1399−162−84 (2-methylbutyryl unit)×2]−, 1053 [1543−162−146 (6-deoxyhexosyl unit)−100 (niloyl unit)−82 (tigloyl unit)]−, 579 [1069−162−146−100−82]−, 417 [579−162]−, and 271 [417−146]− in the negative-ion FAB-MS of 1 (Fig. 2). Taking the J values of signals due to the anomeric and methine protons of the sugar moiety into account, the conformations of the quinovopyranosyl and glucopyranosyl residues were 4C1, and the rhamnopyranosyl residue was 1C4. The absolute configurations of the component 2-methylbutyric acid and nilic acid of the crude resin glycoside fraction of this plant were previously determined to be S and 2S,3S, respectively.13) Therefore, the structure of 1 was defined as 11S-jalapinolic acid 11-O-β-D-glucopyranosyl-(1→3)-O-(2-O-2S,3S-niloyl-4-O-tigloyl)-α-L-rhamnopyranosyl-(1→2)-[O-β-D-glucopyranosyl-(1→6)-O-(3,4-di-O-2S-methylbutyryl)-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside, intramolecular 1,2‴‴-ester, as shown in Fig. 3.
| 1 | 2 | 3 | 4 | |
|---|---|---|---|---|
| Qui-1 | 4.75 d (7.5) | 4.86 d (7.5) | 4.81 d (7.0) | 4.82 d (7.5) |
| 2 | 4.29a) | 4.26a) | 4.29a) | 4.30a) |
| 3 | 4.38 dd (9.0, 9.0) | 4.43 dd (9.0, 9.0) | 4.40a) | 4.40a) |
| 4 | 3.57 dd (9.0, 9.0) | 3.56 dd (9.0, 9.0) | 3.61a) | 3.61 dd (9.0, 9.0) |
| 5 | 3.73 dq (9.0, 6.0) | 3.74 dq (9.0, 6.0) | 3.76a) | 3.77a) |
| 6 | 1.62 d (6.0) | 1.53 d (6.0) | 1.64 d (5.5) | 1.65 d (6.0) |
| Glc-1 | 5.76 d (8.0) | 5.71 d (8.0) | 5.79 d (7.5) | 5.79 d (8.0) |
| 2 | 4.12 dd (8.0, 9.0) | 4.12 dd (8.0, 9.0) | 4.10 dd (7.5, 9.0) | 4.11 dd (8.0, 9.0) |
| 3 | 3.75 dd (9.0, 9.0) | 3.85a) | 3.75 dd (9.0, 9.0) | 3.75 dd (9.0, 9.0) |
| 4 | 3.86 dd (9.0, 9.0) | 3.96 dd (9.0, 9.0) | 3.91a) | 3.93 dd (9.0, 9.0) |
| 5 | 3.66 ddd (2,5, 6.0, 9.0) | 3.64 m | 3.64a) | 3.63 ddd (1.5, 5.0, 9.0) |
| 6 | 4.48 dd (2.5, 11.5) | 4.29a) | 4.29a) | 4.31a) |
| 6 | 4.19 dd (6.0, 11.5) | 4.15a) | 4.16a) | 4.19a) |
| Glc′-1 | 4.87 d (8.0) | 4.94 d (7.5) | 4.87d (8.0) | 4.87 d (7.5) |
| 2 | 3.89 dd (8.0, 9.0) | 3.86a) | 3.85a) | 3.87a) |
| 3 | 5.60 dd (9.0, 9.0) | 5.69 dd (9.5, 9.5) | 5.67 dd (9.5, 9.5) | 5.67 dd (9.5, 9.5) |
| 4 | 5.44 dd (9.0, 9.0) | 5.31 dd (9.5, 9.5) | 5.32 dd (9.5, 9.5) | 5.35 dd (9.5, 9.5) |
| 5 | 3.96a) | 4.28a) | 4.31a) | 4.30a) |
| 6 | 4.13a) | 4.36a) | 4.41a) | 4.39a) |
| 6 | 4.02 dd (4.5, 12.0) | 3.91 br d (11.0) | 3.90a) | 3.93a) |
| Glc″-1 | 5.08 d (8.0) | 5.42 d (8.0) | 5.18 d (7.5) | 5.18 d (7.5) |
| 2 | 3.81 dd (8.0, 9.0) | 4.02 dd (8.0, 9.0) | 3.85a) | 3.87a) |
| 3 | 3.96a) | 3.98a) | 4.03 dd (9.0, 9.0) | 4.03 dd (9.0, 9.0) |
| 4 | 3.91 dd (9.0, 9.0) | 3.95a) | 3.85a) | 3.87a) |
| 5 | 3.95a) | 4.12a) | 4.18a) | 4.20a) |
| 6 | 4.46a) | 4.44a) | 4.53 br d (11.0) | 4.54 dd (1.5, 11.5) |
| 6 | 4.13a) | 4.15a) | 4.13a) | 4.17a) |
| Glc‴-1 | 4.94 d (8.0) | 4.78 d (8.0) | 4.80 d (7.5) | 4.80 d (8.0) |
| 2 | 5.47 dd (8.0, 9.0) | 3.98a) | 4.00 dd (7.5, 9.0) | 4.00 dd (8.0, 9.0) |
| 3 | 4.29a) | 4.17a) | 4.19 dd (9.0, 9.0) | 4.19 dd (9.0, 9.0) |
| 4 | 4.20 dd (9.0, 9.0) | 4.24 dd (9.5, 9.5) | 4.25 dd (9.0, 9.0) | 4.26 dd (9.0, 9.0) |
| 5 | 3.84a) | 3.83a) | 3.84a) | 3.83 ddd (2.0, 5.0, 9.0) |
| 6 | 4.46a) | 4.40a) | 4.42a) | 4.41a) |
| 6 | 4.32 dd (5.0, 11.5) | 4.32a) | 4.32a) | 4.33 dd (5.0, 11.5) |
| Rha-1 | 6.27 d (1.0) | 6.15 s | 6.30 s | 6.31 d (1.0) |
| 2 | 6.15 dd (1.0, 3.5) | 6.06 d (3.0) | 6.09 d (3.0) | 6.10 dd (1.0, 3.5) |
| 3 | 5.17 dd (3.5, 10.0) | 4.97a) | 5.18a) | 5.18 dd (3.5, 10.0) |
| 4 | 5.95 dd (10.0, 10.0) | 4.26a) | 5.92 dd (10.0, 10.0) | 5.93 dd (10.0, 10.0) |
| 5 | 5.25 dq (10.0, 6.5) | 5.03 dq (9.5, 6.0) | 5.17a) | 5.18a) |
| 6 | 1.66 d (6.5) | 1.75 d (6.0) | 1.63 d (6.5) | 1.63 d (6.5) |
| Jla-2 | 2.55a) | 2.35 t (7.5) | 2.34 t (7.5) | 2.52 t (7.5) |
| 2 | 2.41a) | 2.35 t (7.5) | 2.34 t (7.5) | 2.52 t (7.5) |
| 11 | 3.79a) | 3.86a) | 3.84a) | 3.85a) |
| 16 | 0.87 t (7.0) | 0.87 t (7.0) | 0.87 t (7.0) | 0.87 t (7.0) |
| OCH3 | 3.67 s | 3.68 s | ||
| Nla-2 | 2.77 dq (7.0, 7.0) | |||
| 3 | 4.40a) | |||
| 4 | 1.35 d (7.0) | |||
| 5 | 1.30 d (7.0) | |||
| Tig-3 | 7.40 dq (1.0, 7.0) | 7.37 qq (1.0, 6.5) | 7.37 qq (1.0, 7.0) | |
| 4 | 1.75 d (7.0) | 1.74 dd (1.0, 6.5) | 1.74 dd (1.0, 7.0) | |
| 5 | 2.06 br s | 2.06 br s | 2.07 br s | |
| Mba-2 | 2.43 ddq (7.0, 7.0, 7.0) | 2.28 dd (7.0, 7.0, 7.0) | 2.29 ddq (7.0, 7.0, 7.0) | 2.28 ddq (7.0, 7.0, 7.0) |
| 3 | 1.78a) | 1.67a) | 1.67 m | 1.65a) |
| 3 | 1.48a) | 1.37a) | 1.37a) | 1.32a) |
| 4 | 0.94 dd (7.5, 7.5) | 0.90 dd (7.5, 7.5) | 0.86 dd (7.5, 7.5) | 0.85 dd (7.5, 7.5) |
| 5 | 1.17 d (7.0) | 1.10 d (7.0) | 1.10 d (7.0) | 1.10 d (7.0) |
| Mba′-2 | 2.53 ddq (7.0, 7.0, 7.0) | 2.47 ddq (7.0, 7.0, 7.0) | 2.47 ddq (7.0, 7.0, 7.0) | 2.46 ddq (7.0, 7.0, 7.0) |
| 3 | 1.88 m | 1.80a) | 1.80 m | 1.80a) |
| 3 | 1.54 m | 1.50a) | 1.52a) | 1.49a) |
| 4 | 0.97 dd (7.5, 7.5) | 0.94 dd (7.5, 7.5) | 0.94 dd (7.5, 7.5) | 0.93 dd (7.5, 7.5) |
| 5 | 1.22 d (7.0) | 1.19 d (7.0) | 1.19 d (7.0) | 1.18 d (7.0) |
| Mba″-2 | 2.47 ddq (7.0, 7.0, 7.0) | 2.40 ddq (7.0, 7.0, 7.0) | 2.41 ddq (7.0, 7.0, 7.0) | |
| 3 | 1.70a) | 1.75a) | 1.75a) | |
| 3 | 1.34a) | 1.49a) | 1.48a) | |
| 4 | 0.85 dd (7.5, 7.5) | 0.84 dd (7.5, 7.5) | 0.84 dd (7.5, 7.5) | |
| 5 | 1.25 d (7.0) | 1.25 d (7.0) | 1.25 d (7.0) |
δ in ppm from tetramethylsilane (TMS) (coupling constants (J) in Hz are given in parentheses). a) Signals were overlapped with other signals. Qui, quinvopyranosyl; Glc, glucopyranosyl; Rha, rhamnopyranosyl; Jla, jalapinoloyl; Nla, niloyl; Tig, tigloyl; Mba, 2-methylbutyryl.
| 1 | 2 | 3 | 4 | 1 | 2 | 3 | 4 | ||
|---|---|---|---|---|---|---|---|---|---|
| Qui-1 | 103.5 | 102.3 | 102.5 | 102.4 | Jla-1 | 173.2 | 173.9 | 174.0 | 176.1 |
| 2 | 79.2 | 79.5 | 79.2 | 79.2 | 2 | 34.4 | 34.2 | 34.2 | 34.9 |
| 3 | 79.6 | 79.2 | 79.8 | 79.8 | 11 | 82.6 | 80.9 | 81.2 | 81.2 |
| 4 | 76.8 | 77.0 | 77.0 | 77.0 | 16 | 14.2 | 14.3 | 14.2 | 14.2 |
| 5 | 72.6 | 73.0 | 73.0 | 72.7 | OCH3 | 51.3 | 51.3 | ||
| 6 | 18.5 | 18.4 | 18.3 | 18.3 | Nla-1 | 175.3 | |||
| Glc-1 | 101.0 | 101.1 | 101.0 | 101.0 | 2 | 48.4 | |||
| 2 | 74.6 | 75.4 | 74.5 | 74.5 | 3 | 69.1 | |||
| 3 | 89.9 | 89.6 | 89.9 | 89.8 | 4 | 20.6 | |||
| 4 | 70.8 | 70.2 | 70.1 | 70.2 | 5 | 13.2 | |||
| 5 | 77.4 | 76.8 | 76.8 | 76.8 | Tig-1 | 167.6 | 167.6 | 167.6 | |
| 6 | 62.7 | 62.2 | 62.2 | 62.2 | 2 | 129.1 | 129.3 | 129.3 | |
| Glc′-1 | 103.9 | 103.6 | 103.5 | 103.5 | 3 | 138.1 | 137.8 | 137.8 | |
| 2 | 72.5 | 72.4 | 72.2 | 72.2 | 4 | 14.4 | 14.2 | 14.4 | |
| 3 | 75.8 | 75.3 | 75.3 | 75.3 | 5 | 12.8 | 12.8 | 12.8 | |
| 4 | 69.1 | 69.9 | 69.9 | 69.9 | Mba-1 | 175.8 | 175.6 | 175.7 | 175.6 |
| 5 | 73.4 | 73.6 | 73.6 | 73.6 | 2 | 41.1 | 40.9 | 41.0 | 41.0 |
| 6 | 66.7 | 68.4 | 68.4 | 68.4 | 3 | 26.7 | 26.8 | 26.6 | 26.6 |
| Glc″-1 | 105.4 | 105.8 | 105.6 | 105.6 | 4 | 11.8 | 11.6 | 11.7 | 11.6 |
| 2 | 74.8 | 75.7 | 74.9 | 74.9 | 5 | 16.3 | 16.5 | 16.5 | 16.5 |
| 3 | 78.2 | 78.0 | 77.9 | 78.1 | Mba′-1 | 175.8 | 175.5 | 175.6 | 175.6 |
| 4 | 71.6 | 71.8 | 72.0 | 72.0 | 2 | 41.2 | 41.3 | 41.3 | 41.2 |
| 5 | 78.1 | 78.3 | 78.3 | 78.2 | 3 | 27.0 | 27.0 | 27.0 | 27.0 |
| 6 | 63.1 | 62.9 | 63.2 | 63.2 | 4 | 11.8 | 11.8 | 11.8 | 11.8 |
| Glc‴-1 | 102.1 | 104.2 | 104.2 | 104.2 | 5 | 16.6 | 16.7 | 16.8 | 16.8 |
| 2 | 75.0 | 75.3 | 75.4 | 75.4 | Mba″-1 | 176.7 | 176.4 | 176.3 | |
| 3 | 76.0 | 77.9 | 78.1 | 77.9 | 2 | 41.2 | 41.1 | 41.1 | |
| 4 | 71.4 | 71.4 | 71.2 | 71.2 | 3 | 26.6 | 26.6 | 26.6 | |
| 5 | 79.0 | 78.2 | 78.3 | 78.3 | 4 | 11.6 | 11.6 | 11.6 | |
| 6 | 62.2 | 62.4 | 62.4 | 62.4 | 5 | 16.7 | 16.8 | 16.8 | |
| Rha-1 | 97.1 | 97.5 | 96.9 | 96.9 | |||||
| 2 | 73.0 | 72.4 | 72.7 | 72.9 | |||||
| 3 | 75.4 | 78.9 | 74.9 | 74.9 | |||||
| 4 | 74.0 | 73.2 | 74.2 | 74.2 | |||||
| 5 | 67.0 | 69.1 | 67.1 | 67.1 | |||||
| 6 | 18.4 | 18.6 | 18.5 | 18.5 |
δ in ppm from TMS. Qui, quinvopyranosyl; Glc, glucopyranosyl; Rha, rhamnopyranosyl; Jla, jalapinoloyl; Nla, niloyl; Tig, tigloyl; Mba, 2-methylbutyryl.
Compound 2 was obtained as an amorphous powder and exhibited an [M+Na]+ ion peak at m/z 1501 in the positive-ion FAB-MS. The alkaline hydrolysis of 2 yielded 2-methylbutyric acid and 5. The 1H-NMR spectrum of 2 showed signals due to three 2-methylbutyryl residues, one methoxy group, one primary methyl group, two equivalent methylene protons adjacent to a carbonyl group, and six anomeric protons (Table 1). The 13C-NMR spectrum of 2 yielded signals due to 68 carbons, including six anomeric carbons and four ester carbonyl carbons (Table 2). These signals were assigned using 2D-NMR techniques, similar to those used in assigning signals for 1. In addition, 2 was determined to be a non-macrolactone-type resin glycoside. A comparison of the 1H-NMR signals due to the sugar moiety between 2 and 5a indicated acylation shifts (Δδ=δ2–δ5a) of signals due to H-2 (Δδ=ca. 1.01) of Rha and H-3 (Δδ=ca. 1.43) and H-4 (Δδ=ca. 1.43) of Glc′. In addition, the HMBC spectrum of 2, exhibited correlations between the methoxy protons and C-1 of Jla, H-3 of Glc′ and C-1 of Mba, and H-4 of Glc′ and C-1 of Mba′ (Fig. 1). Consequently, the structure of 2 was defined as methyl 11S-jalapinolate 11-O-β-D-glucopyranosyl-(1→3)-O-(2-O-2S-methylbutyryl)-α-L-rhamnopyranosyl-(1→2)-[O-β-D-glucopyranosyl-(1→6)-O-(3,4-di-O-2S-methylbutyryl)-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside, as shown in Fig. 3.
Compound 3 was obtained as an amorphous powder and yielded tiglic acid, 2-methylbutyric acid, and 5 following alkaline hydrolysis. The positive-ion FAB-MS of 3 gave an [M+Na]+ ion peak at m/z 1583, which was 82 mass units lager than that of 2. The molecular formula of 3 was found to be C73H124O35 using HR-positive-ion FAB-MS. The 1H- and 13C-NMR spectra of 3, which were assigned in detail with the aid of 2D-NMR techniques as done for 1, indicated that 3 was composed of 1 mol each of tiglic acid and 5a and 3 mol of 2-methylbutyric acid (Tables 1, 2). In addition, the 1H-NMR spectrum of 3 showed, in comparison with that of 5a, acylation shifts (Δδ=δ3–δ5a) of signals due to H-2 (Δδ=ca. 1.04) and H-4 (Δδ=1.43) of Rha and H-3 (Δδ=ca. 1.41) and H-4 (Δδ=ca. 1.44) of Glc′. From the above data, 3 was assumed to be a derivative of 2 with 1 mol of tiglic acid attached to OH-4 of Rha. This assumption was confirmed using the HMBC spectrum of 3, which revealed a key cross-peak between H-4 of Rha and C-1 of Tig (Fig. 1). Accordingly, the structure of 3 was proposed to be methyl 11S-jalapinolate 11-O-β-D-glucopyranosyl-(1→3)-O-(2-O-2S-methylbutyryl-4-O-tigloyl)-α-L-rhamnopyranosyl-(1→2)-[O-β-D-glucopyranosyl-(1→6)-O-(3,4-di-O-2S-methylbutyryl)-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside, as shown in Fig. 3.
Compound 4 was obtained as an amorphous powder. The negative-ion FAB-MS of 4 exhibited an [M−H]− ion peak at m/z 1545 along with fragment ion peaks at m/z 1463, 1383, 1071, 1053, 579, 417, and 271 (Fig. 2). The molecular formula of 4 was determined to be C72H122O35 using HR-positive-ion FAB-MS. The 1H-NMR spectrum of 4 was superimposable on that of 3 except for the absence of a methoxy group and the chemical shift of the signal due to H2-2 of Jla. Based on these data, 4 was considered to be the free carboxylic acid form of 3. This assumption was confirmed by treating 4 with diazomethane–ether, which yielded 3, and by details of the assignments of NMR signals using the 2D-NMR spectra. Therefore, the structure of 4 was determined to be 11S-jalapinolic acid 11-O-β-D-glucopyranosyl-(1→3)-O-(2-O-2S-methylbutyryl-4-O-tigloyl)-α-L-rhamnopyranosyl-(1→2)-[O-β-D-glucopyranosyl-(1→6)-O-(3,4-di-O-2S-methylbutyryl)-β-D-glucopyranosyl-(1→3)]-O-β-D-glucopyranosyl-(1→2)-β-D-quinovopyranoside, as shown in Fig. 3.
Compounds 1–4 were evaluated for their anti-HSV-1 activity,17) since calysolins I–XIII and soldanelline B previously demonstrated such activity.15,16) The EC50 values of all the compounds were 14 to 54-fold higher than acyclovir, which has been used for the treatment of HSV infections (Table 3). However, all the compounds showed relatively strong cytotoxicity (IC50), and their selectivity index (IC50/EC50) values were ca. 2.0 to 3.9. In addition, the results suggest that the Tig at C-4 of Rha might contribute to the both activities (see, 2 vs. 3). A similar structure–activity relationship was observed in the previous investigation.15) Conversely, cleavage of the macrolactone ring (see, 4 vs. caltsolins VIII (6) and IX (7), which correspond to the macrolactone derivatives of 4)15) and the demethylation of aglycone (see, 3 vs. 4) might reduce both activities. Further, the glucosylation at C-3 of Rha might reduce the selectivity index (see, 1 vs. calysolin II (8)15)). The sort of organic acid unit at C-2 of Rha might not affect the activity (see, 1 vs. 7).
| Sample | EC50 (µg/mL) | IC50 (µg/mL) | IC50/EC50 |
|---|---|---|---|
| 1 | 3.5 | 7.1 | 2.0 |
| 2 | 13.5 | 21.8 | 1.6 |
| 3 | 5.9 | 13.6 | 2.3 |
| 4 | 12.8 | 50.4 | 3.9 |
| 615) | 2.9 | 4.6 | 1.6 |
| 715) | 3.2 | 8.5 | 2.7 |
| 815) | 3.0 | 18.5 | 6.2 |
| Acyclovir | 0.25 | >100 | >400 |
Each value is the mean of duplicate assays.
Our investigation of the fresh leaves, stems, and roots of C. soldanella resulted in the isolation and structural elucidation of four new resin glycosides. They consisted of two types, with one (1) possessing a macrolactone structure similar to calysolins I–XIII, and the others (2–4), with non-macrolactone structures; 2 and 3 were methyl esters of acylated glycosidic acid and 4 was the free carboxylic acid form of 3. It is possible that 2–4 are artifacts formed from the macrolactone resin glycosides during the extraction or isolation or both procedures. However, 3 and 4 were not formed during reflux of 6 and 7, respectively, by using MeOH or a mixture of CHCl3–MeOH–H2O (8 : 2 : 0.2) in the presence of silica gel for 4 h. Furthermore, the two methyl esters of the non-macrolactone resin glycosides, which are similar to 2 and 3, have been previously reported as natural constituents.18) Therefore, 2–4 might be natural constituents. We could not obtain ester-type dimers from this plant.
The instruments and materials used were as cited in the preceding report13,14) unless otherwise specified.
Extraction and IsolationThe cut fresh leaves, stems, and roots of C. soldanella (916.9 g) were extracted with MeOH (10 L) at room temperature for 1 month, and the solvent was removed under reduced pressure to yield a MeOH extract (126.9 g). The MeOH extract was suspended in H2O (3.5 L) and then successively extracted with EtOAc (1.6 L) and BuOH (0.6 L) to yield an EtOAc-soluble fraction (fr.) (48.57 g) and a BuOH-soluble fr. (8.15 g). A part (46.87 g) of the EtOAc-soluble fr. was chromatographed on silica gel column (Merck Art. 1.07734) using gradient mixtures of CHCl3–MeOH–H2O (20 : 1 : 0, 10 : 2 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5, 6 : 4 : 1, 0 : 1 : 0) as eluents to yield fractions (frs.) 1–17. HPLC [column, COSMOSIL 5C18 AR-II, 20 mm i.d.×250 mm (column 1)] of fr. 15 (2.966 g) using 95% MeOH as eluent furnished 7 (273 mg) and frs. 15-1–15-10. Fractions 15-3 (194 mg) and 15-5 (87 mg) were each subjected to HPLC (column 1) eluted with 90% MeOH to yield frs. 13-3-1–15-3-4 from fr. 15-3 and fr. 15-5-1 from fr. 15-5. Fractions 15-3-3 (88 mg) and 15-5-1 (28 mg) were each subjected to HPLC [column, COSMOSIL 5SL-II, 20 mm i.d.×250 mm] eluted with CHCl3–MeOH–H2O (10 : 2 : 0.1 for fr. 15-3-3; 8 : 2 : 0.2 for fr. 15-5-1) to furnish 4 (65 mg) from fr. 15-3-3 and 1 (18 mg) from fr. 15-5-1. Fraction 16 (3.044 g) was subjected to HPLC (column 1) eluted with 90% MeOH to afford frs. 16-1–16-8. HPLC (column 1) of fr. 16-8 (1.973 g) using 90% MeOH as eluent furnished calysolin XI (34 mg), 2 (51 mg), and 3 (27 mg).
1: Amorphous powder. [α]D25 −25.8° (c=1.5, MeOH). Positive-ion FAB-MS m/z: 1567 [M+Na]+. HR-positive-ion FAB-MS m/z: 1567.7491 (Calcd for C72H120O35Na+, 1567.7508). Negative-ion FAB-MS m/z: 1543 [M−H]−, 1443 [1527−100]−, 1399 [1543−144]−, 1069 [1399−162−84×2]−, 1053 [1443−162−82−146]−, 579 [1053−144−84×2−162]−, 417 [579−162]−, 271 [417−146]−. 1H-NMR spectral data: see Table 1. 13C-NMR spectral data: see Table 2.
2: Amorphous powder. [α]D25 −12.3° (c=1.1, MeOH). Positive-ion FAB-MS m/z: 1501 [M+Na]+. HR-positive-ion FAB-MS m/z: 1501.7410 (Calcd for C68H118O34Na+, 1501.7402). 1H-NMR spectral data: see Table 1. 13C-NMR spectral data: see Table 2.
3: Amorphous powder. [α]D25 −14.5° (c=1.9, MeOH). Positive-ion FAB-MS m/z: 1583 [M+Na]+. HR-positive-ion FAB-MS m/z: 1583.7859 (Calcd for C73H124O35Na+, 1583.7821). 1H-NMR spectral data: see Table 1. 13C-NMR spectral data: see Table 2.
4: Amorphous powder. [α]D19 −14.6° (c=1.2, MeOH). Positive-ion FAB-MS m/z: 1569 [M+Na]+. HR-positive-ion FAB-MS m/z: 1569.7673 (Calcd for C72H122O35Na+, 1569.7664). Negative-ion FAB-MS m/z: 1545 [M−H]−, 1463 [1545−82]−, 1383 [1545−162]−, 1071 [1383−146−84−82]−, 1053 [1383−162−84×2]–, 579 [1053−162−146−84−82]−, 417 [579−162]–, 271 [417−146]−. 1H-NMR spectral data: see Table 1. 13C-NMR spectral data: see Table 2.
Alkaline Hydrolysis of 1–4Solutions of 1 (5 mg), 2 (7 mg), 3 (7 mg), and 4 (5 mg) in 1 M KOH–1,4-dioxane (1 : 1, 2 mL) were each heated at 95°C for 1 h. The reaction mixture was adjusted to pH 3 with 1 M HCl and then diluted with H2O (10 mL) and extracted with ether (3×5 mL). The ether layer was dried over MgSO4 and evaporated to furnish an organic acid fr., which was analyzed by using GC [column, Unisole 30T (5%), 3.2 mm i.d.×2.0 m glass column (column 2); carrier gas N2, 1.0 kg/cm2; column temperature, 120°C; tR (min): 4.42 (2-methylbutyric acid) for 1–4, 10.38 (tiglic acid) for 1, 3, and 4]. A part of the organic acid fr. was methylated with diazomethane–ether and then the reaction mixture was analyzed by GC [column, column 2; column temperature, 100°C; carrier gas, N2 1.2 kg/cm2; tR (min): 4.21 (methyl nilate) for 1].
The aqueous layer was desalted by MCI gel CHP 20 column chromatography (H2O, acetone) to give a glycosidic acid as an amorphous powder (2 mg from 1, 5 mg from 2, 4 mg from 3, 3 mg from 4). All of the glycosidic acids derived from 1–4 were identical with calysolic acid C by comparison of 1H-NMR spectrum with that of authentic sample.11)
Methylation of 4Compound 4 (3 mg) in MeOH (1 mL) was treated with diazomethane–ether. The reaction mixture was evaporated in vacuo to give 4a (3 mg), which was identified as 3 by comparison of 1H-NMR spectrum with that of authentic sample.
Reflux of 6 and 7 with MeOH or a Mixture of CHCl3–MeOH–H2OCompounds 6 (1 mg) and 7 (1 mg) were dissolved in MeOH (1 mL) or a mixture of CHCl3–MeOH–H2O (8 : 2 : 0.2, 1 mL) in the presence of silica gel (Merck Art. 1.07734, 50 mg), and each refluxed for 4 h. Compounds 2–4 were not detected using TLC analysis (plate, Merck TLC silica gel 60 RP-18 F254S; solvent, 95% MeOH) of both refluxed mixtures.
The authors declare no conflict of interest.
