Notes
Triterpene Glycosides from the Stems and Leaves of Lonicera japonica
2014 Volume 62 Issue 1 Pages 92-96
Details
2014 Volume 62 Issue 1 Pages 92-96
Five new triterpene glycosides, namely lonicerosides F–J (1–5), together with five known ones, were isolated from the stems and leaves of Lonicera japonica. Based on extensive spectroscopic analysis, including two-dimensional (2D)-NMR experiments, and the results of hydrolysis, the structures of the new compounds were determined to be 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-β-D-glucopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester (loniceroside F), 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester (loniceroside G), 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(4-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester (loniceroside H), 3β-[(α-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester (loniceroside I), and 3β-[(α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl)oxy]-olean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (loniceroside J).
Lonicera japonica THUNBERG (Caprifoliaceae) is native to East Asia, including P. R. China, Japan and Korea. L. japonica is traditionally used as a medicinal plant,1) and the dried stems and leaves of this plant are listed in the Japanese Pharmacopoeia XVI under the crude drug name Nindou. The stems and leaves of L. japonica have been chemically investigated, and iridoid glycosides and a number of triterpene glycosides have been isolated; some of these compounds may contribute to the medicinal effects of this crude drug.2–6) As part of our continuing investigation of triterpene glycosides from medicinal plants,7–21) we conducted phytochemical screening of the stems and leaves of L. japonica and isolated five new triterpene glycosides, together with five known ones. Here we report the structures of these compounds, based on extensive spectroscopic analysis, including two-dimensional (2D)-NMR experiments, and the results of hydrolysis.
The stems and leaves of L. japonica were extracted with hot MeOH. The concentrated MeOH extract was passed through a porous-polymer polystyrene resin (Diaion HP-20) column, eluted with 30% MeOH, EtOH, and EtOAc. The EtOH eluate, in which triterpene glycosides were enriched, was subjected to a series of chromatographic separations, resulting in the isolation of compounds 1 (12.3 mg), 2 (126 mg), 3 (6.7 mg), 4 (163 mg), 5 (14.1 mg), 6 (735 mg), 7 (127 mg), 8 (783 mg), 9 (12.1 mg), and 10 (141 mg) (Fig. 1). The structures of the known compounds were identified as 3β-[(α-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (6, loniceroside A),2) 3β-[(α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (7, loniceroside B),2) 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (8, loniceroside C),3) 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-β-D-glucopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (9, loniceroside D),22) and 3β-[(β-D-glucopyranosyl)oxy]-olean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester (10, loniceroside E).22)
Loniceroside F (1) was obtained as an amorphous solid. Its molecular formula was determined as C54H88O23 on the basis of the high resolution (HR)-electrospray ionization (ESI)-time-of-flight (TOF)-MS data (m/z: 1127.5635 [M+Na]+). The IR spectrum suggested the presence of hydroxy (3370 cm−1) and carbonyl (1743 cm−1) groups. The 1H-NMR spectrum of 1 showed signals for six tertiary methyl groups at δH 1.20, 1.11, 0.97, 0.95, 0.86, and 0.83 (each s), and a trisubstituted olefinic proton at δH 5.43 (br s). In addition, signals for four anomeric protons were observed at δH 6.56 (br s), 6.11 (d, J=8.1 Hz), 5.13 (d, J=7.8 Hz), and 4.99 (d, J=7.8 Hz). The three-proton doublet signal at δH 1.77 (J=6.2 Hz) indicated the presence of one deoxyhexopyranosyl unit in 1. Acid hydrolysis of 1 with 0.2 M HCl in dioxane–H2O (1 : 1) produced an aglycone (1a), identified as 3β,23-dihydroxyolean-12-en-28-oic acid (hederagenin),23) as well as L-rhamnose and D-glucose as the carbohydrate moieties. The monosaccharides were identified by direct HPLC analysis of the hydrolysate, which was performed on an aminopropyl-bonded silica gel column using MeCN–H2O (17 : 3) as the solvent system. Detection used both refractive index (RI) and optical rotation (OR). In the 13C-NMR spectrum of 1, the C-3 and C-28 carbons of the aglycone moiety were observed at δC 82.4 and 176.5, respectively, suggesting that 1 is a 3,28-bisdesmoside. Through an 1H–1H correlation spectroscopy (COSY) experiment on 1, we sequentially assigned H-1 to H2-6 and Me-6 of the monosaccharides. Their signal splitting patterns and coupling constants (Table 1) indicated the presence of three β-D-glucopyranosyl (4C1) units and an α-L-rhamnopyranosyl unit in 1. The heteronuclear multiple quantum coherence (HMQC) and heteronuclear single quantum coherence (HSQC)-total correlation spectroscopy (TOCSY) spectra of 1 were used to associate the protons with the corresponding one-bond coupled carbon resonances. The carbon chemical shifts thus assigned were compared with those of the reference methyl glycosides,24) taking into account the known effects of known O-glycosylation; as a result, 1 appeared to contain two terminal β-D-glucopyranosyl units (Glc(II) and Glc(III)), a 2,6-disubstituted β-D-glucopyranosyl unit (Glc(I)), and a terminal α-L-rhamnopyranosyl unit (Rha). In the heteronuclear multiple bond connectivity (HMBC) spectrum of 1, long-range correlations were observed between H-1 of Glc(I) at δH 5.13 and C-3 of the aglycone at δC 82.4, H-1 of Rha at δH 6.56 and C-2 of Glc(II) at δC 75.3, H-1 of Glc(III) at δH 4.99 and C-6 of Glc(II) at δC 69.4, and H-1 of Glc(II) at δH 6.11 and C-28 of the aglycone at δC 176.5. Accordingly, 1 was established as 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-β-D-glucopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester.
| 1 | 2 | 3 | 4 | 5 | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Position | δH | J (Hz) | Position | δH | J (Hz) | Position | δH | J (Hz) | Position | δH | J (Hz) | Position | δH | J (Hz) | |||||
| Glc(I) | 1′ | 5.13 d | 7.8 | Glc(I) | 1′ | 5.13 d | 7.8 | Glc(I) | 1′ | 5.14 d | 7.8 | Ara | 1′ | 4.98 d | 7.2 | Glc(I) | 1′ | 4.92 d | 7.2 |
| 2′ | 4.05 dd | 8.8, 7.8 | 2′ | 4.04 dd | 8.3, 7.8 | 2′ | 4.04 dd | 8.9, 7.8 | 2′ | 4.43 dd | 8.8, 7.2 | 2′ | 4.29 dd | 8.6, 7.2 | |||||
| 3′ | 4.19 dd | 9.0, 8.8 | 3′ | 4.20 dd | 8.8, 8.3 | 3′ | 4.20 dd | 8.9, 8.9 | 3′ | 4.09 dd | 8.8, 3.3 | 3′ | 4.30 dd | 8.8, 8.6 | |||||
| 4′ | 4.26 dd | 9.0, 9.0 | 4′ | 4.25 dd | 9.4, 8.8 | 4′ | 4.26 ddd | 9.0, 8.9 | 4′ | 4.26 br s | 4′ | 4.17 dd | 9.5, 8.8 | ||||||
| 5′ | 3.91 m | 5′ | 3.90 ddd | 9.4, 4.8, 2.9 | 5′ | 3.90 ddd | 9.0, 5.0, 2.6 | 5′a | 4.27 br d | 10.9 | 5′ | 3.94 ddd | 9.5, 5.3, 2.8 | ||||||
| 6′a | 4.52 br d | 12.6 | 6′a | 4.50 br d | 11.7 | 6′a | 4.51 dd | 11.9, 2.6 | b | 3.72 br d | 10.9 | 6′a | 4.54 dd | 11.8, 5.3 | |||||
| b | 4.41 br d | 12.6 | b | 4.40 dd | 11.7, 4.8 | b | 4.40 dd | 11.9, 5.0 | b | 4.37 dd | 11.8, 2.8 | ||||||||
| Glc(II) | 1″ | 6.11 d | 8.1 | Glc(II) | 1″ | 6.11 d | 8.1 | Glc(II) | 1″ | 6.11 d | 8.1 | Glc | 1″ | 6.12 d | 8.0 | Rha(I) | 1″ | 6.57 br s | |
| 2″ | 4.40 dd | 8.6, 8.1 | 2″ | 4.34 dd | 8.8, 8.1 | 2″ | 4.38 dd | 8.7, 8.1 | 2″ | 4.34 dd | 8.3, 8.0 | 2″ | 4.87 br d | 3.1 | |||||
| 3″ | 4.27 dd | 8.6, 8.3 | 3″ | 4.25 dd | 9.4, 8.8 | 3″ | 4.26 dd | 8.9, 8.7 | 3″ | 4.25 dd | 9.5, 8.3 | 3″ | 4.69 dd | 9.3, 3.1 | |||||
| 4″ | 4.30 dd | 8.7, 8.3 | 4″ | 4.30 dd | 9.4, 8.8 | 4″ | 4.24 dd | 8.9, 8.3 | 4″ | 4.30 dd | 9.5, 9.2 | 4″ | 4.35 dd | 9.3, 9.3 | |||||
| 5″ | 4.08 m | 5″ | 4.00 m | m | 5″ | 4.07 | m | 5″ | 4.04 ddd | 9.2, 4.2, 1.7 | 5″ | 4.79 dq | 9.3, 6.2 | ||||||
| 6″a | 4.67 br d | 10.1 | 6″a | 4.63 br d | 10.6 | 6″a | 4.61 br d | 11.4 | 6″a | 4.64 dd | 11.2, 1.7 | 6″ | 1.71 d | 6.2 | |||||
| b | 4.29 br d | 10.1 | b | 4.25 br d | 10.6 | b | 4.29 br d | 11.4 | b | 4.25 dd | 11.2, 4.2 | ||||||||
| Rha | 1‴ | 6.56 br s | Rha | 1‴ | 6.51 br s | Rha | 1‴ | 6.54 br s | Rha | 1‴ | 6.51 d | 1.2 | Glc(II) | 1‴ | 6.13 d | 8.1 | |||
| 2‴ | 4.79 br d | 3.2 | 2‴ | 4.78 br d | 3.0 | 2‴ | 4.79 br d | 3.3 | 2‴ | 4.78 dd | 3.3, 1.2 | 2‴ | 4.41 dd | 8.6, 8.1 | |||||
| 3‴ | 4.56 dd | 9.2, 3.2 | 3‴ | 4.54 dd | 9.5, 3.0 | 3‴ | 4.55 dd | 9.3, 3.3 | 3‴ | 4.54 dd | 9.3, 3.3 | 3‴ | 4.28 dd | 8.6, 8.6 | |||||
| 4‴ | 4.35 dd | 9.3, 9.2 | 4‴ | 4.33 dd | 9.5, 9.1 | 4‴ | 4.34 dd | 9.4, 9.3 | 4‴ | 4.33 dd | 9.4, 9.3 | 4‴ | 4.32 dd | 9.3, 8.6 | |||||
| 5‴ | 4.54 dq | 9.3, 6.2 | 5‴ | 4.51 dq | 9.1, 6.0 | 5‴ | 4.53 dq | 9.4, 6.1 | 5‴ | 4.51 dq | 9.4, 6.1 | 5‴ | 4.08 ddd | 9.3, 4.4, 2.3 | |||||
| 6‴ | 1.77 d | 6.2 | 6‴ | 1.74 d | 6.0 | 6‴ | 1.76 d | 6.1 | 6‴ | 1.74 d | 6.1 | 6‴a | 4.64 dd | 12.2, 2.3 | |||||
| b | 4.29 dd | 12.2, 4.4 | |||||||||||||||||
| Glc(III) | 1″″ | 4.99 d | 7.8 | Xyl | 1″″ | 4.86 d | 7.4 | Xyl | 1″″ | 4.96 d | 7.5 | Xyl | 1″″ | 4.86 d | 7.5 | Rha(II) | 1″″ | 6.58 br s | |
| 2″″ | 3.99 dd | 8.8, 7.8 | 2″″ | 3.96 dd | 9.1, 7.4 | 2″″ | 4.25 dd | 8.6, 7.5 | 2″″ | 3.97 dd | 9.2, 7.5 | 2″″ | 4.79 br d | 3.1 | |||||
| 3″″ | 4.19 dd | 9.1, 8.8 | 3″″ | 5.64 dd | 9.2, 9.1 | 3″″ | 3.99 dd | 9.5, 8.6 | 3″″ | 5.64 dd | 9.2, 9.2 | 3″″ | 4.55 dd | 9.1, 3.1 | |||||
| 4″″ | 4.22 | 4″″ | 4.15 ddd | 9.9, 9.2, 5.6 | 4″″ | 5.35 ddd | 9.6, 9.5, 5.4 | 4″″ | 4.16 ddd | 9.8, 9.2, 5.4 | 4″″ | 4.35 dd | 9.2, 9.1 | ||||||
| 5″″ | 3.91 m | 5″″a | 4.27 dd | 10.4, 5.6 | 5″″a | 4.29 dd | 11.4, 5.4 | 5″″a | 4.27 dd | 10.9, 5.4 | 5″″ | 4.54 dq | 9.2, 6.1 | ||||||
| 6″″a | 4.47 dd | 11.8, 2.1 | b | 3.61 dd | 10.4, 9.9 | b | 3.52 dd | 11.4, 9.6 | b | 3.61 dd | 10.9, 9.8 | 6″″ | 1.77 d | 6.1 | |||||
| b | 4.36 br d | 11.8 | |||||||||||||||||
| Ac | 2.00 s | Ac | 1.97 s | Ac | 2.00 s | Xyl | 1″‴ | 4.89 d | 7.4 | ||||||||||
| 2″‴ | 3.96 dd | 8.5, 7.4 | |||||||||||||||||
| 3″‴ | 4.11 dd | 8.9, 8.5 | |||||||||||||||||
| 4″‴ | 4.17 ddd | 9.4, 8.9, 4.5 | |||||||||||||||||
| 5″‴a | 4.30 dd | 10.8, 4.5 | |||||||||||||||||
| b | 3.64 dd | 10.8, 9.4 | |||||||||||||||||
Lonicerosides G (2) and H (3), which had the same molecular formula of C55H88O23, were found to each contain an acetyl group by examination of their 1H- and 13C-NMR spectra [δH 2.00 (3H, s), δC 170.6 (C=O) and 21.2 (Me) in 2; δH 1.97 (3H, s), δC 170.5 (C=O) and 20.9 (Me) in 3]. Alkaline hydrolysis of 2 and 3 with 10% KOH (dioxane–H2O, 1 : 1) gave the same hydrolysate (9). When the 1H-NMR spectra of 2 and 3 were compared with that of 9, H-3 of the xylopyranosyl unit (Xyl) was shifted to a lower field by 1.54 ppm in 2, while H-4 of Xyl was moved downfield by 1.18 ppm in 3. Furthermore, in the HMBC spectra of 2 and 3, the carbonyl carbon of the acetyl group of 2 at δC 170.6 showed a long-range correlation with H-3 of Xyl at δH 5.64 (dd, J=9.2, 9.1 Hz), and that of 3 at δC 170.5 showed a correlation with H-4 at δH 5.35 (ddd, J=9.6, 9.5, 5.4 Hz). Thus, 2 and 3 were respectively assigned the following structures: 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester and 3β-[(β-D-glucopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(4-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester.
Loniceroside I (4) had a molecular formula of C54H86O22, as determined by HR-ESI-TOF-MS. The presence of an acetyl group in 4 was shown by the 1H- and 13C-NMR spectra [δH 2.00 (3H, s), δC 170.6 (C=O) and 21.2 (Me)]. Alkaline hydrolysis of 4 furnished hydrolysate 6, indicating that 4 was a monoacetate of 6. In the HMBC spectrum of 4, a correlation peak was observed between H-3 of Xyl at δH 5.64 and the acetyl carbonyl carbon at δC 170.6. Accordingly, 4 was assigned as 3β-[(α-L-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid O-(3-O-acetyl-β-D-xylopyranosyl)-(1→6)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl ester.
Loniceroside J (5) was obtained as an amorphous solid and had a molecular formula of C59H96O25, as determined by HR-ESI-TOF-MS. The deduced molecular formula was larger than that of 10, with the difference corresponding to C6H10O4, indicating the presence of an additional deoxyhexosyl unit in 5. The 1H-NMR spectrum showed signals for five anomeric protons at δH 6.58 (br s), 6.57 (br s), 6.13 (d, J=8.1 Hz), 4.92 (d, J=7.2 Hz), and 4.89 (d, J=7.4 Hz), together with signals for seven tertiary methyl groups at δH 1.24, 1.19, 1.14, 1.05, 0.91, and 0.87×2 (each s). Acid hydrolysis of 5 with 1 M HCl yielded oleanolic acid, D-glucose, L-rhamnose, and D-xylose. Comparing the whole 13C-NMR spectrum of 5 with that of 10, we observed the signals due to the aglycone moiety and the triglycoside residue linked to C-28 of the aglycone at almost same positions in the two compounds. However, a set of six additional signals corresponding to a terminal α-L-rhamnopyranosyl moiety appeared at δC 101.7, 72.4, 72.5, 74.1, 69.6, and 18.7, and the signal due to C-2 of the glucopyranosyl unit attached to C-3 of the aglycone was shifted downfield by 2.0 ppm and observed at δ 77.8, suggesting that the C-2 position of the glucosyl moiety (Glc(I)) was glycosylated by the additional L-rhamnosyl unit (Rha(I)). In the HMBC spectrum of 5, H-1 of Rha(I) at δH 6.57 showed a long-range correlation with C-2 of Glc(I) at δC 77.8, whereas H-1 of Glc(I) at δH 4.92 was correlated to C-3 of the aglycone at δC 88.9. A diglycoside consisting of a rhamnosyl-(1→2)-glucosyl unit was found to be attached to C-3 of the aglycone. Furthermore, the triglycoside attached to C-28 of the aglycone was confirmed to be the same as that of 10 by the analysis of HMBC spectrum. Thus, Thus, 5 was designated as 3β-[(α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl)oxy]-olean-12-en-28-oic acid O-α-L-rhamnopyranosyl-(1→2)-[β-D-xylopyranosyl-(1→6)]-β-D-glucopyranosyl ester.
In conclusion, we have identified 1–5 as new triterpene bisdesmosides, and isolated 9 and 10 from the stems and leaves of L. japonica for the first time. Compounds 6 and 8, which were obtained in good yield and have not been isolated from other plants, are possible marker compounds for identifying L. japonica.
Optical rotations were measured using a JASCO P-1030 (Tokyo, Japan) automatic digital polarimeter. IR spectra were recorded on a JASCO FT-IR 620 spectrophotometer. NMR spectra were recorded on a Bruker DRX-500 spectrometer (500 MHz for 1H-NMR, Karlsruhe, Germany) and and a Bruker AV-600 spectrometer (600 MHz for 1H-NMR) using standard Bruker pulse programs. Chemical shifts are given as δ values referenced to tetramethylsilane as an internal standard. ESI-TOF-MS data were obtained on a Waters-Micromass LCT (Manchester, U.K.) mass spectrometer. Porous-polymer polystyrene resin (Diaion HP-20, Mitsubishi-Chemical, Tokyo, Japan), silica gel (300 mesh, Fuji-Silysia Chemical, Aichi, Japan), ODS silica gel (75 µm, Nacalai-Tesque, Kyoto, Japan), and Sephadex LH-20 (GE Healthcare Japan, Tokyo, Japan) were used for column chromatography. TLC was carried out on plates precoated with Silica gel 60 F254 (0.25 mm thick, Merck, Darmstadt, Germany) or RP-18 F254S (0.25 mm thick, Merck), and plates were developed by spraying with 10% H2SO4 aq. solution followed by heating. HPLC was performed by using a system comprised of a CCPM pump (Tosoh, Tokyo, Japan), a CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh) or a Shodex OR-2 detector (Showa-Denko, Tokyo, Japan), and a Rheodyne injection port.
The stems and leaves of L. japonica (lot. no. 312323) were purchased from Uchida Wakanyaku Co., Ltd. (Tokyo, Japan) in September 2006. A voucher specimen has been deposited in our laboratory (voucher No. 07-09-LJSL, Department of Medicinal Pharmacognosy).
The dried stems and leaves of L. japonica (5.0 kg) were extracted with hot MeOH (24 L). The MeOH extract was concentrated under reduced pressure, and the viscous concentrate (535 g) was passed through a Diaion HP-20 column [30% MeOH (6 L), EtOH (6 L), and EtOAc (9 L)]. The EtOH eluate (fraction B, 130 g) was chromatographed on silica gel eluted with CHCl3–MeOH gradients (9 : 1, 4 : 1, 2 : 1), and finally with MeOH alone, to afford nine fractions (B-i–B-ix). Fraction B-i was subjected to column chromatography on ODS silica gel and eluted with MeOH–H2O (7 : 3) to give 5 (163 mg). Fraction B-ii was subjected to column chromatography on ODS silica gel eluted with MeOH–H2O (3 : 2, 7 : 3) to give two subfractions (B-ii-a and B-ii-b). Fraction B-ii-a was subjected to a silica gel column eluted with CHCl3–MeOH–H2O (20 : 10 : 1, 7 : 4 : 1) and preparative HPLC using MeCN–H2O (2 : 5) to afford 2 (126 mg) and 3 (6.7 mg). Column chromatography of fraction B-iii on octadecyl silica (ODS) silica gel eluted with MeOH–H2O (3 : 2, 7 : 3) gave 6 (735 mg), 8 (783 mg), and 10 (141 mg). Fraction B-iv was chromatographed on ODS silica gel eluted with MeOH–H2O (13 : 7) to give three fractions (B-iv-a to B-iv-c). Fraction B-iv-a was subjected to silica gel column chromatography eluted with CHCl3–MeOH–H2O (20 : 10 : 1), to ODS silica gel column chromatography eluted with MeOH–H2O (13 : 7), and to preparative HPLC using MeCN–H2O (16 : 37) to give 1 (12.3 mg) and 9 (12.1 mg). Fraction B-iv-b was chromatographed on ODS silica gel eluted with MeOH–H2O (3 : 2) to yield 7 (127 mg). Fraction B-iv-c was chromatographed on a silica gel column eluted with CHCl3–MeOH–H2O (20 : 10 : 1) and a Sephadex LH-20 column eluted with MeOH to give 5 (14.1 mg).
Loniceroside F (1): Amorphous solid. [α]D26 －41.8 (c=0.10, MeOH). HR-ESI-TOF-MS m/z: 1127.5635 [M+Na]+ (Calcd for C54H88NaO23: 1127.5613). IR νmax (film) cm−1: 3370 (OH), 2937 and 2878 (CH), 1743 (C=O); 1H-NMR (C5D5N) δH: 5.43 (1H, br s, H-12), 4.31 (1H, d, J=10.6 Hz, H-23a), 3.63 (1H, d, J=10.6 Hz, H-23b), 1.20 (3H, s, Me-27), 1.11 (3H, s, Me-26), 0.97 (3H, s, Me-25), 0.95 (3H, s, Me-24), 0.86 (3H, s, Me-30), 0.83 (3H, s, Me-29); 1H-NMR signals for the sugar moieties: see Table 1; 13C-NMR (C5D5N) δ: 38.8, 25.8, 82.4, 43.4, 47.8, 18.3, 32.3, 39.9, 48.2, 37.0, 23.8, 122.5, 144.3, 42.3, 28.7, 23.4, 47.2, 41.9, 46.4, 30.7, 34.0, 33.0, 64.9, 13.6, 16.3, 17.5, 25.8, 176.5, 33.1, and 23.8 (C-1–C-30), 105.8, 75.9, 78.7, 71.6, 78.4, and 62.8 (C-1′–C-6′), 94.8, 75.3, 79.5, 71.2, 77.7, and 69.4 (C-1″–C-6″), 101.4, 72.2, 72.6, 73.8, 69.8, and 18.7 (C-1‴–C-6‴), 105.3, 75.2, 78.3, 71.5, 78.3, and 62.6 (C-1″″–C-6″″).
Compound 1 (2.5 mg) was dissolved in 0.2 M HCl (dioxane–H2O, 1 : 1, 2.0 mL) and heated at 95°C for 2 h under an Ar atmosphere. After H2O (5 mL) was added, the solution was extracted with EtOAc (10 mL×2). The extract was concentrated and chromatographed on silica gel eluted with CHCl3–MeOH (19 : 1) to yield hederagenin (1.1 mg). The aqueous portion was neutralized by passage through an Amberlite IRA-96SB column (Organo, Tokyo, Japan) and chromatographed on Diaion HP-20 eluted with H2O to yield a sugar fraction (1.4 mg). The sugar fraction was dissolved in H2O (1 mL), passed through a Sep-Pak C18 cartridge (Waters, Milford, MA, U.S.A.), and then analyzed by HPLC under the following conditions: column, Capcell Pak NH2 UG80 (4.6 mm i.d.×250 mm, 5 µm, Shiseido, Japan); solvent, MeCN–H2O (17 : 3); detection, RI and OR; flow rate, 1.0 mL/min. HPLC analysis of the sugar fraction showed the presence of L-rhamnose and D-glucose; tR (min) 8.87 (L-rhamnose, negative optical rotation), 17.00 (D-glucose, positive optical rotation).
Loniceroside G (2): Amorphous solid. [α]D27 －27.2 (c=0.10, MeOH). HR-ESI-TOF-MS m/z: 1117.5803 [M+H]+ (Calcd for C55H89O23: 1117.5795). IR νmax (film) cm−1: 3374 (OH), 2940 (CH), 1730 (C=O); 1H-NMR (C5D5N) δH: 5.44 (1H, br s, H-12), 4.31 (1H, d, J=10.7 Hz, H-23a), 3.63 (1H, d, J=10.7 Hz, H-23b), 1.20 (3H, s, Me-27), 1.11 (3H, s, Me-26), 0.98 (3H, s, Me-25), 0.95 (3H, s, Me-24), 0.90 (3H, s, Me-30), 0.84 (3H, s, Me-29); 1H-NMR signals for the sugar moieties: see Table 1; 13C-NMR (C5D5N) δ: 38.7, 25.8, 82.3, 43.4, 47.7, 18.3, 32.3, 39.9, 48.1, 36.9, 23.8, 122.7, 144.1, 42.2, 28.6, 23.4, 47.1, 41.9, 46.3, 30.7, 34.0, 32.9, 64.8, 13.6, 16.2, 17.5, 25.9, 176.6, 33.1, and 23.8 (C-1–C-30), 106.6, 75.8, 78.6, 71.5, 78.3, and 62.7 (C-1′–C-6′), 94.8, 75.5, 79.6, 70.9, 77.4, and 69.2 (C-1″–C-6″), 101.4, 72.1, 72.5, 73.8, 69.8, and 18.7 (C-1‴–C-6‴), 105.4, 72.4, 78.6, 69.0, and 66.7 (C-1″″–C-5″″), 21.2 and 170.6 (acetyl).
Loniceroside H (3): Amorphous solid. [α]D23 －13.7 (c=0.10, MeOH). HR-ESI-TOF-MS m/z: 1139.5603 [M+Na]+ (Calcd for C55H88NaO23: 1139.5614). IR νmax (film) cm−1: 3362 (OH), 2937 (CH), 1739 (C=O); 1H-NMR (C5D5N) δH: 5.42 (1H, br s, H-12), 4.33 (1H, d, J=10.7 Hz, H-23a), 3.64 (1H, d, J=10.7 Hz, H-23b), 1.21 (3H, s, Me-27), 1.11 (3H, s, Me-26), 0.99 (3H, s, Me-25), 0.97 (3H, s, Me-24), 0.93 (3H, s, Me-30), 0.87 (3H, s, Me-29); 1H-NMR signals for the sugar moieties: see Table 1; 13C-NMR (C5D5N) δ: 38.8, 25.9, 82.4, 43.4, 47.7, 18.3, 32.4, 40.0, 48.2, 37.0, 23.8, 122.7, 144.1, 42.3, 28.6, 23.4, 47.2, 42.0, 46.3, 30.7, 34.0, 33.0, 64.8, 13.6, 16.3, 17.5, 25.9, 176.5, 33.1, and 23.8 (C-1–C-30), 105.8, 75.9, 78.7, 71.6, 78.3, and 62.7 (C-1′–C-6′), 94.7, 75.5, 79.6, 71.0, 77.8, and 68.7 (C-1″–C-6″), 101.4, 72.2, 72.6, 73.8, 69.8, and 18.8 (C-1‴–C-6‴), 105.5, 74.7, 75.0, 73.0, and 63.3 (C-1″″–C-5″″), 20.9 and 170.5 (acetyl).
Compounds 2 and 3 (each 5.0 mg) were hydrolyzed with 10% KOH (dioxane–H2O, 1 : 1, 1.0 mL) for 90 min at room temperature. The reaction mixture was neutralized by passing through an Amberlite IRB (Organo) column and chromatographed on silica gel eluted with CHCl3–MeOH–H2O (7 : 4 : 1) to give 9 (2.5 mg from 2; 2.8 mg from 3).
Loniceroside I (4): Amorphous solid. [α]D26 －15.0 (c=0.10, MeOH). HR-ESI-TOF-MS m/z: 1087.5767 [M+H]+ (Calcd for C54H87O22: 1087.5789). IR νmax (film) cm−1: 3404 (OH), 2941 (CH), 1729 (C=O); 1H-NMR (C5D5N) δH: 5.45 (1H, br s, H-12), 4.26 (1H, d, J=10.4 Hz, H-23a), 3.62 (1H, d, J=10.4 Hz, H-23b), 1.19 (3H, s, Me-27), 1.11 (3H, s, Me-26), 1.01 (3H, s, Me-25), 0.91 (3H, s, Me-30), 0.90 (3H, s, Me-24), 0.85 (3H, s, Me-29); 1H-NMR signals for the sugar moieties: see Table 1; 13C-NMR (C5D5N) δ: 38.9, 26.1, 82.0, 43.4, 47.7, 18.2, 32.3, 40.0, 48.2, 36.9, 23.9, 122.7, 144.1, 42.2, 28.6, 23.4, 47.1, 41.9, 46.3, 30.7, 34.0, 32.9, 64.5, 13.6, 16.2, 17.5, 25.9, 176.6, 33.1, and 23.8 (C-1–C-30), 106.6, 73.1, 74.7, 69.6, and 66.9 (C-1′–C-5′), 94.8, 75.5, 79.6, 70.9, 77.4, and 69.2 (C-1″–C-6″), 101.5, 72.2, 72.5, 73.8, 69.8, and 18.8 (C-1‴–C-6‴), 105.5, 72.4, 78.7, 69.0, and 66.7 (C-1″″–C-5″″), 21.2 and 170.6 (acetyl).
Compound 4 (5.0 mg) was alkaline hydrolyzed as described for 2 and 3 to give 6 (2.8 mg).
Loniceroside J (5): Amorphous solid. [α]D23 －15.7 (c=0.10, MeOH). HR-ESI-TOF-MS m/z: 1227.6122 [M+Na]+ (Calcd for C59H96NaO25: 1227.6137). IR νmax (film) cm−1: 3359 (OH), 2927 (CH), 1736 (C=O); 1H-NMR (C5D5N) δH: 5.41 (1H, br s, H-12), 1.24 (3H, s, Me-27), 1.19 (3H, s, Me-23), 1.14 (3H, s, Me-24), 1.05 (3H, s, Me-26), 0.91 (3H, s, Me-30), 0.87 (3H×2, each s, Me-25 and Me-29); 1H-NMR signals for the sugar moieties: see Table 1; 13C-NMR (C5D5N) δ: 39.0, 26.8, 88.9, 39.4, 56.1, 18.6, 32.3, 79.9, 48.0, 36.9, 23.7, 122.7, 144.1, 42.2, 28.7, 23.3, 47.2, 41.9, 46.4, 30.7, 34.0, 33.1, 28.1, 17.1, 15.7, 17.4, 25.9, 176.6, 33.1, and 23.8 (C-1–C-30), 105.4, 77.8, 79.9, 72.1, 78.1, and 62.8 (C-1′–C-6′), 101.7, 72.4, 72.5, 74.1, 69.6, and 18.7 (C-1″–C-6″), 94.7, 75.3, 79.6, 71.1, 77.6, and 69.0 (C-1‴–C-6‴), 101.4, 72.2, 72.5, 73.8, 69.7, and 18.7 (C-1″″–C-6″″), 105.5, 74.8, 78.0, 71.0, and 67.0 (C-1″‴–C-5″‴).
A solution of 5 (5.0 mg) was subjected to acidic hydrolysis as described for 1 to yield an aglycone fraction (1.1 mg) and a sugar fraction (2.1 mg). The aglycone fraction was chromatographed on silica gel eluted with CHCl3–MeOH (19 : 1) to give olaenolic acid (0.2 mg). HPLC analysis of the sugar fraction under the same conditions as in the case of 1 showed the presence of L-rhamnose, D-xylose, and D-glucose; tR (min) 8.48 (L-rhamnose, negative optical rotation), 10.84 (D-xylose, positive optical rotation), 18.59 (D-glucose, positive optical rotation).
