Notes
Structures of Ryobunins A–C from the Leaves of Clethra barbinervis
2013 Volume 61 Issue 5 Pages 581-586
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2013 Volume 61 Issue 5 Pages 581-586
From a MeOH extract of the leaves of Clethra barbinervis Sieb. et Zucc., ryobunins A–C, three new triterpene glucosides, i.e. one ursane, one seco-ursane and one oleanane-type glucoside, along with four known compounds were isolated. Their structures were elucidated based on chemical and spectral evidence.
About 40 species of the genus Clethra, belonging to the family Clethraceae, are distributed in Asia and America, with only one species, Clethra (C.) barbinervis Sieb. et Zucc. (Japanese name: ryo-bu), growing wild in Japan from the southern part of Hokkaido to Kyusyu. C. barbinervis is a deciduous tree that grows to about 4–7 m in height, the young leaves of which were used as a foodstuff in famines.1,2) Phytochemical studies on this plant have been reported for the isolation of several triterpenoids.3,4) Further investigation of the constituents of the leaves of this plant has led to the isolation of three new triterpene glucosides, named ryobunins A–C. The structures of the new compounds were elucidated on the basis of spectral and physicochemical evidence.
Isolation and Structure ElucidationAir-dried and finely chopped leaves of C. barbinervis (6.28 kg) were extracted with MeOH at room temperature and the concentrated MeOH extract was partitioned with solvents of increasing polarity. The 1-BuOH-soluble fraction was separated by column chromatography (CC) on a highly-porous synthetic resin (Diaion HP-20), normal and reversed-phase (ODS: octadecyl silanized) silica gel CC, and droplet counter-current chromatography (DCCC) to afford three new triterpene glucosides (1–3), ryobunins A (1, 0.00015%), B (2, 0.00030%), and C (3, 0.00046%), along with four known compounds, clethric acid 28-O-β-d-glucopyranosyl ester5) (4, 0.00015%), kakisaponin A6) (5, 0.00052%), barbinervic acid3,7) (6, 0.00041%), and clethric acid8) (7, 0.00036%) (Chart 1). Their structures were elucidated by means of extensive spectroscopic techniques and a chemical method, mainly one and two-dimensional NMR spectroscopy, and electrospray ionization (ESI)-mass spectrometry (MS), and acid hydrolysis, respectively.
Ryobunin A (1) was isolated as an amorphous powder with negative optical rotation ([α]D21 −21.5 in MeOH). The IR spectrum of 1 showed absorption bands at 3377, 1733 and 1072 cm−1 ascribable to hydroxy, carbonyl and ether functional groups, respectively. The molecular formula, C36H58O11, of 1 was determined by high-resolution-electrospray ionization (HR-ESI)-MS analysis. The 1H-NMR (pyridine-d5) spectrum of 1 showed five singlet methyl signals at δH 1.01, 1.13, 1.43, 1.54 and 1.74, one doublet methyl signal at δ 1.05 (J=6.6 Hz), one set of methylene signals attached to a hydroxy group at δH 3.79 and 4.04 (each 1H, both d, J=11.0 Hz), two methine signals attached to a hydroxy functional group at δH 4.38 (br t, J=2.7 Hz) and 5.10 (m), and an anomeric proton signal at δH 6.19 (d, J=8.1 Hz). The 13C-NMR spectrum exhibited six methyl signals (δC 16.0, 16.7, 17.2, 23.4, 25.5, 26.1) (Table 1), one methylene signal attached to a hydroxy group (δC 65.7), two methine signals attached to hydroxy groups (δC 70.0, 73.0), one anomeric carbon signal (δC 96.0), and one ester carbonyl carbon signal (δC 175.3). The NMR chemical shifts of 1 were similar to those of barbinervic acid (6), which was isolated from the title plant,6) except for the presence of a glucopyranose moiety and an extra hydroxy group. The presence and absolute configuration of d-glucose were confirmed by HPLC analysis of an acid hydrolyzate of 1 with an optical rotation detector, and the position of the hydroxy group was determined to be at C-16 from the 1H–1H correlation spectroscopy (COSY) from H2-15 (δH 1.67, 2.62) to H-16 (δH 5.10), and the heteronuclear multiple-bond correlations (HMBC) between H3-27 (δH 1.74) and C-15 (δC 36.8), and H-18 (δH 3.30) and C-16 (δC 73.0) (Fig. 1). Therefore, the structure of the aglycone of 1 was deduced to be 16-hydroxybarbinervic acid. The geometry of the hydroxy group was determined be in an α-orientation by comparison with 13C-NMR data reported for 16-α (δC ca. 74)9) and 16-β (δC ca. 64)10) hydroxy derivatives of similar compounds (Table 1). The carbon skeleton and the positions of three remaining functional groups were confirmed by the HMBC experiment, which showed long-range correlations between the following protons and carbons: H-18 and C-13, 17 and 28; H3-23 and C-3, 4, 5 and 24; H2-24 and C-3, 4, 5 and 23; H3-29 and C-19 and 20; and H3-30 and C-19, 20 and 21. The HMBC cross peak of the anomeric proton [δH 6.19 (d, 8.1 Hz)] with C-28 (δC 175.3) indicated the location of the glucopyranosyl unit to be at the C-28 position of the aglycone and its β-linkage was concluded from the J value (d, J=8.1 Hz) of the anomeric proton signal. The ester linkage between C-28 and C-1′ was also corroborated by the upfield chemical shift of the anomeric carbon. Therefore, compound 1 was assigned as shown in Chart 1.
Dual arrow curve denotes HMBC correlations were observed in both directions.
| 1 | 2 | 3 | ||||
|---|---|---|---|---|---|---|
| 1 | 33.9 | 1.42 (m) | 34.3 | 1.54 (m) | 33.8 | 1.31 (m) |
| 1.79 (m) | 1.98 (m) | 1.79 (m) | ||||
| 2 | 26.3 | 1.66 (m) | 26.4 | 1.80 (m) | 26.3 | 1.15 (m) |
| 2.09 (m) | 2.08 (m) | 1.81 (m) | ||||
| 3 | 70.0 | 4.38 (br t, J=2.7 Hz) | 70.1 | 4.40 (br t, J=2.5 Hz) | 70.0 | 4.37 (br t, J=2.5 Hz) |
| 4 | 43.8 | — | 44.0 | — | 43.8 | — |
| 5 | 50.0 | 1.82 (br d, J=12.0 Hz) | 50.3 | 1.92 (br d, J=11.9 Hz) | 50.2 | 1.83 (br d, J=12.7 Hz) |
| 6 | 19.1 | 1.00 (m) | 18.9 | 1.39 (br d, J=11.4 Hz) | 19.2 | 1.04 (m) |
| 1.60 (m) | 1.67 (m) | 1.64 (m) | ||||
| 7 | 34.1 | 1.40 (m) | 35.6 | 1.40 (dt, J=13.3, 2.8 Hz) | 33.8 | 1.40 (m) |
| 1.49 (br d, J=13.4 Hz) | 1.49 (dd, J=13.3, 3.6 Hz) | 1.53 (m) | ||||
| 8 | 40.9 | — | 41.5 | — | 40.3 | — |
| 9 | 46.8 | 2.06 (m) | 45.8 | 2.34 (dd, J=13.0, 2.6 Hz) | 48.3 | 2.03 (m) |
| 10 | 37.4 | — | 37.5 | — | 37.6 | — |
| 11 | 24.1 | 2.06–2.14 (2H, m) | 30.3 | 1.52 (m) | 24.3 | 2.06 (2H, m) |
| 1.85 (m) | ||||||
| 12 | 129.4 | 5.67 (br s) | 74.1 | 4.47 (br t, J=3.6 Hz) | 124.0 | 5.49 (br s) |
| 13 | 137.1 | — | 147.1 | — | 143.4 | — |
| 14 | 41.1 | — | 43.5 | — | 42.2 | — |
| 15 | 36.8 | 1.67 (m) | 28.4 | 1.50 (dd, J=14.5, 2.8 Hz) | 29.0 | 1.19 (br d, J=14.2 Hz) |
| 2.62 (dd, J=15.3, 3.7 Hz) | 2.45 (br d, J=14.6 Hz) | 2.19 (dd, J=14.2, 2.8 Hz) | ||||
| 16 | 73.0 | 5.10 (m) | 29.2 | 1.17 (m) | 30.7 | 2.41 (br d, J=12.7 Hz) |
| 2.16 (dt, J=14.3, 3.6 Hz) | 2.82 (br d, J=12.7 Hz) | |||||
| 17 | 51.1 | — | 47.7 | — | 46.4 | — |
| 18 | 54.5 | 3.30 (br s) | 128.1 | 5.90 (s) | 44.1 | 3.60 (br s) |
| 19 | 71.4 | — | 211.9 | — | 83.9 | 3.73 (d, J=2.8 Hz) |
| 20 | 41.7 | 1.40 (m) | 47.5 | 2.40 (dd, J=13.3, 6.7 Hz) | 39.0 | — |
| 21 | 28.7 | 1.09 (m) | 27.6 | 1.08 (m) | 75.1 | 3.80 (dd, J=5.0, 2.7 Hz) |
| 2.26 (dd, J=13.6, 4.3 Hz) | 1.33 (br d, J=10.9 Hz) | |||||
| 22 | 35.8 | 1.94 (dd, J=13.0, 5.3 Hz) | 38.7 | 1.54 (m) | 40.0 | 2.33 (dd, J=14.6, 2.7 Hz) |
| 2.35 (br d, J=13.6 Hz) | 1.83 (m) | 2.45 (dd, J=14.6, 5.0 Hz) | ||||
| 23 | 23.4 | 1.54 (s) | 23.4 | 1.57 (s) | 23.5 | 1.53 (s) |
| 24 | 65.7 | 3.79 (d, J=11.0 Hz) | 65.7 | 3.77 (d, J=10.9 Hz) | 65.7 | 3.79 (d, J=11.0 Hz) |
| 4.04 (d, J=11.0 Hz) | 4.04 (d, J=10.9 Hz) | 4.03 (d, J=11.0 Hz) | ||||
| 25 | 16.0 | 1.01 (s) | 17.0 | 0.90 (s) | 15.9 | 0.97 (s) |
| 26 | 17.2 | 1.13 (s) | 18.2 | 1.01 (s) | 17.5 | 1.11 (s) |
| 27 | 25.5 | 1.74 (s) | 23.6 | 1.46 (s) | 24.1 | 1.46 (s) |
| 28 | 175.3 | — | 174.8 | 176.9 | ||
| 29 | 26.1 | 1.43 (s) | 27.8 | 2.07 (s) | 23.8 | 1.40 (s) |
| 30 | 16.7 | 1.05 (d, J=6.6 Hz) | 16.2 | 0.98 (d, J=6.6 Hz) | 26.0 | 0.99 (s) |
| Glc | ||||||
| 1′ | 96.0 | 6.19 (d, J=8.1 Hz) | 96.1 | 6.26 (d, J=7.8 Hz) | 96.1 | 6.26 (d, J=7.8 Hz) |
| 2′ | 73.9 | 4.12 (m) | 74.1 | 4.12 (dd, J=8.4, 7.8 Hz) | 74.1 | 4.11 (dd, J=8.5, 7.8 Hz) |
| 3′ | 78.7 | 4.25 (m) | 78.7 | 4.19 (dd, J=9.0, 8.4 Hz) | 78.8 | 4.23 (br d, J=9.0 Hz) |
| 4′ | 71.2 | 4.25 (m) | 71.3 | 4.22 (dd, J=9.0, 9.0 Hz) | 71.0 | 4.22 (dd, J=9.1, 9.0 Hz) |
| 5′ | 79.2 | 4.01 (m) | 79.1 | 3.98 (ddd, J=9.0, 4.5, 2.5 Hz) | 79.2 | 3.97 (ddd, J=9.1, 4.5, 2.4 Hz) |
| 6′ | 62.3 | 4.34 (dd, J=11.9, 4.9 Hz) | 62.4 | 4.27 (dd, J=11.5, 4.5 Hz) | 62.2 | 4.28 (dd, J=12.0, 4.5 Hz) |
| 4.44 (dd, J=11.9, 2.4 Hz) | 4.38 (dd, J=11.5, 2.5 Hz) | 4.37 (dd, J=12.0, 2.4 Hz) | ||||
Ryobunin B (2) was also isolated as an amorphous powder exhibiting negative optical rotation ([α]D20 −10.3 in MeOH). The molecular formula, C36H58O11, indicating eight degrees of unsaturation, of 2 was determined by positive-ion HR-ESI-MS. Its IR spectrum showed absorptions at 3381, 1732, 1700 and 1073 cm−1 ascribable to hydroxy, two carbonyl and ether functional groups, respectively. Acid hydrolysis of 2 liberated d-glucose, which was identified by HPLC analysis. Among the 36 13C-NMR signals, those for one terminal glucopyranosyl ester unit (δC 96.1, 74.1, 78.7, 71.3, 79.1, 62.4) were observed (Table 1). The remaining 30 signals comprised those of six methyls (δC 16.2, 17.0, 18.2, 23.4, 23.6, 27.8), nine methylenes (δC 18.9, 26.4, 27.6, 28.4, 29.2, 30.3, 34.3, 35.6, 38.7), three methines (δC 45.8, 47.5, 50.3), five quaternary carbons (δC 37.5, 41.5, 43.5, 44.0, 47.7), together with one trisubstituted double bond (δC 128.1, 147.1), two carbonyl carbons (δC 174.8, 211.9), one oxymethylene carbon (δC 65.7), and two oxymethine carbons (δC 70.1, 74.1). The 1H-NMR spectrum of 2 showed four singlet methyl signals at δH 0.90, 1.01, 1.46 and 1.57, one doublet methyl signal at δH 0.98 (J=6.6 Hz), and a fairly deshielded singlet methyl signal at δH 2.07. The proton sequence from H3-30 (δH 0.98) to H2-22 through H-20 and H2-21 was observed on the 1H–1H COSY spectrum (Fig. 2). The fairly deshielded methyl signal (H3-29) showed correlation cross peaks with C-19 (δC 211.9) and C-20 (δC 47.5), and H3-30 with C-20 and C-21 in the HMBC spectrum (Fig. 2). Together with further HMBC correlations from H2-21 to C-17 and H2-22 to C-28, the presence of a 2-keto-3-methylpentyl moiety was confirmed, its connection point being determined to be at C-17. HMBC correlation peaks of the olefinic proton (δH 5.90) to C-12 (δC 74.1), C-13 (δC 147.1), C-17 (δC 47.7), and C-22 (δC 38.7) placed the hydroxy group at the C-12 position, and the double bond between the C-13 and C-18 positions. A typical signal for an equatorial proton [δH 4.47 (br t, J=3.6 Hz)], attached to a hydroxylated carbon at C-12, indicated the hydroxy group was in an α-orientation, which was supported by the significant nuclear Overhauser effect spectroscopy (NOESY) correlation between H-12 and H-18 (Fig. 3). This conclusion was also supported that β-ilexanolic acid which has the C-12 hydroxy group in a β-orientation showed a large coupling constant between H-11 and H-12.11) The remaining two hydroxy groups were placed at the same positions as those of 1 from the close resemblance of the NMR spectral data for their A-rings (Table 1). The above findings indicated that the aglycone was a tetracyclic seco-triterpene, formed through cleavage of the C-18 and C-19 bond. The configuration at the C-20 position is probably the same as that of the possible precursor, ursane. This was further supported a positive Cotton effect at 284 nm in the circular dichroism (CD) spectrum.11–13) The mode of glucoside linkage was β, as judged from the coupling constant (J=7.8 Hz) of the anomeric proton on 1H-NMR spectroscopy, and in the HMBC spectrum, the anomeric proton (δH 6.26) showed a cross peak with an ester carbonyl carbon (δC 174.8). Accordingly, the structure of 2 was determined to be as shown in Chart 1.
Dual arrow curves denote HMBC correlations were observed in both directions.
Sugar moiety is omitted for clarity.
Ryobunin C (3) was also isolated as an amorphous powder exhibiting negative optical rotation. The molecular formula was also the same as that of compounds 1 and 2. The one- and two-dimensional NMR spectra showed signals corresponding to six methyls [δH 0.97, 0.99, 1.11, 1.40, 1.46, 1.53 (each s, H3-25, 29, 26, 30, 27, 23, respectively)], one set of oxymethylene protons [δH 3.79, 4.03 (both d, J=11.0 Hz, H2-24)], three oxymethine protons [δH 4.37 (br t, J=2.5 Hz, H-3), 3.73 (d, J=2.8 Hz, H-19), 3.80 (dd, 5.0, 2.7 Hz, H-21)], an olefinic proton [δH 5.49 (br s, H-12)], and an anomeric proton [δH 6.26 (d, J=7.8 Hz, H-1′)] (Table 1). The olefinic carbon signals at δC 124.0 and 143.4 along with other carbon signals observed on 13C-NMR led to the identification of the aglycone as an oleanolic acid derivative. Since the NMR spectroscopic data for the A-, B- and C-rings were superimposable on those of 1, two hydroxy groups must be on the D- and/or E-ring. The characteristic signal of H-18 in the 1H-NMR spectrum appeared at δH 3.60 as a broad singlet, instead of a double of doublet, showing that one of the protons of C-19 was replaced by a hydroxy group. The hydroxy functional groups at the C-19 and C-21 positions were substantiated by the HMQC and HMBC experiments, the latter of which showed long-range correlations between the following protons and carbons: H3-29 and C-19 and 20; and H3-30 and C-19, 20 and 21 (Fig. 4). The stereochemistry at C-19 and C-21 was established on the basis of coupling constants of the oxygenated methine protons, H-19 [δH 3.73 (d, J=2.8 Hz)] and H-21 [δH 3.80 (dd, J=5.0, 2.7 Hz)], it being assumed that the hydroxy groups of C-19 and 21 were both in axial-like α-orientations (Fig. 5). The NOESY correlations observed between H-19 and H3-29 and 30, and H-21 and H3-29 and 30 supported this assumption, namely these two hydrogens, respectively, bisect the two methyl groups (Fig. 5). The presence and absolute configuration of d-glucose were confirmed by HPLC analysis of an acid hydrolyzate of 3, and the anomeric configuration of the sugar unit was determined to be β from the coupling constant of the anomeric proton signal. Furthermore, the cross peak observed between δH 6.26 (H-1′) and δC 176.9 (C-28) indicated the attachment of a β-d-glucopyranosyl unit at C-28. Consequently, the structure of 3 was elucidated to be as shown in Chart 1. Since ryobunin C (3) was obtained enough amount for further experiment, the alkaline hydrolysis of 3 was conducted to give a corresponding aglycone (3a), whose physic-chemical data were included in the text.
Dual arrow curves denote HMBC correlations were observed in both directions.
Sugar moiety is omitted for clarity.
In conclusion, an ursane-type triterpene glucoside, ryobunin A (1), a seco-ursane type triterpene glucoside, ryobunin B (2), and an oleanane type triterpene glucoside, ryobunin C (3), together with clethric acid 28-O-β-d-glucopyranosyl ester (4) kakisaponin A (5), barbinervic acid (6), and clethric acid (7) were isolated from the leaves of C. barbinervis collected in Hiroshima prefecture. β-Ilexanolic acid (C-18–C-19-seco-ursane) glycoside, whose aglycone is similar to that of ryobunin B (2), have been isolated from Ilex crenata by Kakuno et al.11,12) Although the isolation of several 21,22-dihydroxyoleanane-type triterpenes and their glycosides has been reported from different plant families, mainly from the Aceraceae14) and Camelliaceae,15) a 19,21-dihydroxyoleanane-type one like the aglycone (3a) of ryobunin C (3) was reported here for the first time.
The following instruments were used to obtain physical and spectroscopic data. CD: JASCO J-720 spectroplarimeter; specific rotations: JASCO P-1030 polarimeter; IR spectra: Shimadzu FT-710 spectrometer; HR-ESI mass spectra: LTQ Orbitrap XL; and 1H- and 13C-NMR spectra: JEOL ECA-600 spectrometer at 600 MHz and 150 MHz, respectively, with tetramethylsilane as an internal standard. A highly-porous synthetic resin, Diaion HP-20, was purchased from Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Silica gel CC was performed on silica gel 60 [(E. Merck, Darmstadt, Germany), 70–230 mesh]. Reversed-phase ODS open CC (RPCC) was performed on Cosmosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) [Φ=50 mm, L=25 cm, linear gradient: MeOH–H2O]. HPLC was performed on an ODS-3 column (Inertsil; GL Science, Tokyo, Japan; Φ=10 mm, L=25 cm, flow rate: 2.00 mL/min), and the eluate was monitored with a refractive index monitor, RID-6A (Shimadzu, Kyoto, Japan). Precoated silica gel 60 F254 plates (E. Merck; 0.25 mm in thickness) were used for TLC analyses, visualized by spraying with a 10% solution of H2SO4 in ethanol and heated to around 150°C on a hotplate.
Plant MaterialThe leaves of C. barbinervis were collected in Hiroshima prefecture in 2011. A voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University (11-CB-Hiroshima-0726).
Isolation of Compounds 1‒7Air-dried leaves of C. barbinervis (6.28 kg, Hiroshima) were extracted three times with MeOH (15 L×3) at room temperature for one week and then concentrated to 3 L in vacuo. The concentrated extract was washed with n-hexane (3 L, 72.3 g) and then the MeOH layer was concentrated to a gummy mass. The latter was suspended in water (3 L) and then extracted with EtOAc (3 L) to give 123.6 g of an EtOAc-soluble fraction. The aqueous layer was extracted with 1-BuOH (3 L) to give a 1-BuOH-soluble fraction (133 g), and the remaining water-layer was concentrated to furnish 289 g of a water-soluble fraction. The 1-BuOH-soluble fraction (130 g) was applied to a Diaion HP-20 column {2.0 kg, MeOH–H2O [1 : 4 (9 L)→2 : 3 (9 L)→3 : 2 (9 L)→4 : 1 (9 L)]→MeOH (10 L)} to give seven fractions [Fr. 1 (15.7 g), Fr. 2 (44.8 g), Fr. 3 (46.7 g), Fr. 4 (8.1 g), Fr. 5 (7.0 g), Fr. 6 (4.0 g), and Fr. 7 (4.0 g)]. Fraction 4 (8.1 g) and Fr. 5 (7.0 g) were subjected to normal-phase silica gel CC [0.5 kg, CHCl3 (6 L)→CHCl3–MeOH {[49 : 1 (6 L)→19 : 1 (6 L)→9 : 1 (6 L)→17 : 5 (6 L)→4 : 1 (6 L)→3 : 1 (6 L)→7 : 3 (6 L)]→CHCl3–MeOH–H2O (15 : 6 : 1) (6 L)→MeOH (6 L)} to give 14 fractions [Fr. 1 (946 mg), Fr. 2 (713 mg), Fr. 3 (982 mg), Fr. 4 (797 mg), Fr. 5 (484 mg), Fr. 6 (509 mg), Fr. 7 (837 mg), Fr. 8 (480 mg), Fr. 9 (447 mg), Fr. 10 (46.3 mg), Fr. 11 (546 mg), Fr. 12 (381 mg), Fr. 13 (267 mg), and Fr. 14 (4.5 g)]. Fraction 4,5-3 (982 mg) and Fr. 4,5-4 (797 mg) were separated by reversed-phase silica gel CC [120 g, MeOH–H2O (1 : 9→1 : 4→3 : 7→2 : 3→1 : 1→3 : 2→7 : 3→4 : 1)→MeOH] to yield 16 fractions [Fr. 4,5-3,4-1 (174 mg), Fr. 4,5-3,4-2 (828 mg), Fr. 4,5-3,4-3 (99.6 mg), Fr. 4,5-3,4-4 (48.0 mg), Fr. 4,5-3,4-5 (102 mg), Fr. 4,5-3,4-6 (49.9 mg), Fr. 4,5-3,4-7 (29.0 mg), Fr. 4,5-3,4-8 (29.5 mg), Fr. 4,5-3,4-9 (16.2 mg), Fr. 4,5-3,4-10 (7.5 mg), Fr. 4,5-3,4-11 (9.8 mg), Fr. 4,5-3,4-12 (7.4 mg), Fr. 4,5-3,4-13 (6.9 mg), Fr. 4,5-3,4-14 (2.2 mg), Fr. 4,5-3,4-15 (200 mg), and Fr. 4,5-3,4-16 (9.8 mg)]. Fraction 4,5-3,4-15 (200 mg) was purified by HPLC [MeOH–acetone–H2O (25 : 20 : 55, v/v)] to give kakisaponin A (5, 33.4 mg) from the peak at 96 min. Fraction 4,5-5 (484 mg) and Fr. 4,5-6 (509 mg) were separated by reversed-phase silica gel CC [120 g, MeOH–H2O (1 : 9→1 : 4→3 : 7→2 : 3→1 : 1→3 : 2→7 : 3→4 : 1)→MeOH] to yield eight fractions [Fr. 4,5-5,6-1 (68.6 mg), Fr. 4,5-5,6-2 (27.3 mg), Fr. 4,5-5,6-3 (53.7 mg), Fr. 4,5-5,6-4 (166 mg), Fr. 4,5-5,6-5 (281 mg), Fr. 4,5-5,6-6 (281 mg), Fr. 4,5-5,6-7 (146 mg), and Fr. 4,5-5,6-8 (19.3 mg)]. Fraction 4,5-5,6-6 (281 mg) was purified by HPLC [MeOH–H2O (3 : 2, v/v)] to give clethric acid 28-O-β-d-glucopyranosyl ester (4, 18.0 mg) and ryobunin C (3, 27.5 mg) from the peaks at 23 min and 30 min, respectively. Fraction 4,5-5,6-7 (145.5 mg) was purified by HPLC [MeOH–H2O (3 : 2, v/v)] to give ryobunin A (1, 8.5 mg) and ryobunin B (2, 8.5 mg) from the peaks at 35 min and 45 min, respectively. Fraction 6 (4.0 g) was separated by reversed-phase silica gel CC [120 g, MeOH–H2O (1 : 9→1 : 4→3 : 7→2 : 3→1 : 1→3 : 2→7 : 3→4 : 1)→MeOH] to yield nine fractions [Fr. 6-1 (77.2 mg), Fr. 6-2 (104 mg), Fr. 6-3 (382 mg), Fr. 6-4 (370 mg), Fr. 6-5 (213 mg), Fr. 6-6 (459 mg), Fr. 6-7 (330 mg), Fr. 6-8 (235 mg), and Fr. 6-9 (1.09 g)]. Fraction 6-6 (459 mg) was purified by HPLC [MeOH–acetone–H2O (3 : 3 : 4, v/v/v)] to give clethric acid (7, 21.8 mg) and barbinervic acid (6, 24.5 mg) from the peaks at 50 min and 67 min, respectively.
The known compounds were identified by comparison of their physical data ([α]D, IR, MS, 1H-, and 13C-NMR) with reported values.
Ryobunin A (1): Amorphous powder; [α]D21 −21.5 (c=0.82, MeOH); IR (film) νmax 3377, 2931, 1733, 1649, 1455, 1072, 1028 cm−1; 1H-NMR (600 MHz, pyridine-d5) δH: given in Table 1; 13C-NMR (150 MHz, pyridine-d5) δC: given in Table 1; HR-ESI-MS (positive-ion mode) m/z: 689.3867 [M+Na]+ (Calcd for C36H58O11Na: 689.3871).
Ryobunin B (2): Amorphous powder; [α]D20 −10.3 (c=1.51, MeOH); IR (film) νmax 3381, 2936, 1732, 1700, 1649, 1455, 1073, 1029 cm−1; 1H-NMR (600 MHz, pyridine-d5) δH: given in Table 1; 13C-NMR (150 MHz, pyridine-d5) δC: given in Table 1; CD: [θ]284 +72.6 (c=3.0×10−3 m, MeOH); MeOH, HR-ESI-MS (positive-ion mode) m/z: 689.3867 [M+Na]+ (Calcd for C36H58O11Na: 689.3871).
Ryobunin C (3): Amorphous powder; [α]D21 −2.1 (c=2.75, MeOH); IR (film) νmax 3395, 2934, 1732, 1649, 1455, 1072, 1031 cm−1; 1H-NMR (600 MHz, pyridine-d5) δH: given in Table 1; 13C-NMR (150 MHz, pyridine-d5) δC: given in Table 1; HR-ESI-MS (positive-ion mode) m/z: 689.3882 [M+Na]+ (Calcd for C36H58O11Na: 689.3871).
Acid Hydrolysis of Ryobunins A–C (1–3)A solution of 1–3 (1 mg) in 1 m HCl (1.0 mL) was heated under reflux for 3 h. After cooling, the reaction mixture was neutralized with Amberlite IRA-400 (OH− form), and the resin was removed by filtration. Then, the filtrate was extracted with EtOAc. The aqueous layer was subjected to HPLC analysis [column: Shodex Asahipak NH 2P-50 4E, 250×4.6 mm i.d.; mobile phase: MeCN–H2O (3 : 1, v/v); detection: optical rotation (JASCO 2090Plus Chiral); flow rate: 1.0 mL/min] to identify d-glucose, which was confirmed by comparison of its retention time with that of the authentic sample; tR: 6.2 min (d-glucose, positive optical rotation).
Alkaline Hydrolysis of Ryobunin C (3)A solution of ryobunin C (3) (8.2 mg) in 10% aqueous KOH–50% aqueous 1,4-dioxane (1 : 1, 1.0 mL) was stirred at 60°C for 3 h. The reaction mixture was neutralized with DOWEX HCR W2 (H+ form) and then the resin was removed by filtration. The filtrate was extracted with EtOAc and the EtOAc layer was evaporated under vacuum to give a 3a (4.0 mg). Compound 3a: Amorphous powder, [α]D22 +12.5 (c=0.20, MeOH); IR (film) νmax 3275, 2919, 1689, 1650, 1540, 1455, 1203, 1023 cm−1; 1H-NMR (600 MHz, pyridine-d5) δ: 5.62 (1H, br s, H-12), 4.46 (1H, t, J=2.5 Hz, H-3), 4.10 (1H, d, J=10.9 Hz, H-24a), 4.00 (1H, dd, J=5.0, 3.3 Hz, H-21), 3.79 (1H, br s, H-18), 3.87 (1H, d, J=10.9 Hz, H-24b), 3.84 (1H, d, J=2.5 Hz, H-19), 2.91 (1H, br dd, J=13.6, 3.4 Hz, H-16), 2.63 (1H, dd, J=14.2, 5.0 Hz, H-22a), 2.53 (1H, dd, J=14.2, 3.3 Hz, H-22b), 2.49 (1H, br d, J=13.6 Hz, H-16), 2.15 (1H, m, H-10), 2.13 (1H, m, H-2a), 2.08 (3H, m, H2-11, H-15), 1.92 (1H, br d, J=12.0 Hz, H-5), 1.87 (1H, dd, J=13.2, 2.6 Hz, H-1a), 1.84 (1H, br d, J=13.0 Hz, H-2b), 1.75 (1H, br d, J=12.2 Hz, H-6a), 1.68 (1H, m, H-7a), 1.60 (1H, m, H-6b), 1.42 (1H, br d, J=13.2 Hz, H-1b), 1.42 (1H, m, H-7b), 1.63 (3H, s, H3-23), 1.57 (3H, s, H3-27), 1.52 (3H, s, H3-29), 1.28 (1H, dd, J=13.7, 2.8 Hz, H-15), 1.19 (3H, s, H3-30), 1.11 (3H, s, H3-26), 1.03 (3H, s, H3-25); 13C-NMR (150 MHz, pyridine-d5) δC: 180.5 (C-28), 144.2 (C-13), 123.2 (C-12), 84.3 (C-19), 75.4 (C-21), 70.0 (C-3), 65.7 (C-24), 50.3 (C-5), 48.4 (C-9), 46.2 (C-17), 44.2 (C-18), 44.0 (C-4), 42.4 (C-14), 41.1 (C-22), 40.1 (C-8), 39.4 (C-20), 37.7 (C-10), 34.1 (C-1), 33.8 (C-7), 31.2 (C-16), 29.1 (C-15), 26.5 (C-2), 26.3 (C-30), 24.4 (C-11), 24.0 (C-27), 23.8 (C-29), 23.6 (C-23), 19.2 (C-6), 15.9 (C-25), 17.5 (C-26); HR-ESI-MS (positive-ion mode) m/z: 527.3340 [M+Na]+ (Calcd for C30H48O6Na: 527.3343).
