Novel Steroidal Glycosides from the Whole Plants of Helleborus foetidus

Abstract

Phytochemical analysis of the whole Helleborus foetidus plants identified 28 steroidal glycosides (1–28), including 20 novel spirostanol glycosides (1–20) and a novel furostanol glycoside (21). The structures of the newly identified compounds were elucidated by two-dimensional NMR spectroscopy and hydrolytic cleavage. Compounds 12, 13, and 15 were determined to be spirostanol trisdesmosides bearing sugar moieties at the C-1, -21, and -24 hydroxy groups of the aglycone unit. The isolated compounds were subsequently evaluated for cytotoxic activity against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells. In particular, 7 showed cytotoxic activity against the HL-60 and A549 cells, with IC₅₀ values of 5.9 and 6.6 µM, respectively, whereas 19 was selectively cytotoxic to A549 cells with an IC₅₀ value of 5.5 µM.

Introduction

Plants from the genus Helleborus are perennials belonging to the Ranunculaceae family, and are widely distributed throughout Europe.¹⁾ Helleborus species are poisonous plants and a series of bufadienolides have been isolated from H. caucasicus,^2,3) H. odorus,^4,5) H. thibetanus,^6–8) and H. torquatus.⁹⁾ Recently, various bufadienolides from H. orientalis and H. foetidus were isolated and identified, some of which showed significantly potent cytotoxic activity and induced apoptosis via a mitochondria dependent pathway in cultured tumor cells.^10,11) Because Helleborus species has been reported to concomitantly contain the glycoside derivatives of C₂₇ steroids (spirostanols and furostanols),^12–18) further phytochemical examination of the methanolic extract of H. foetidus was performed using whole plants with a specific focus on steroidal glycosides. The isolation of 28 steroidal glycosides (1–28), including 20 novel spirostanol glycosides (1–20) and a novel furostanol glycoside (21) was achieved. The structures of the newly identified compounds were determined from their two-dimensional NMR spectra and hydrolytic cleavage. The cytotoxic activities of the isolated compounds were evaluated against HL-60 human promyelocytic leukemia cells and A549 human lung carcinoma cells.

Results and Discussion

Whole plants of H. foetidus (fresh weight, 3.3 kg) were extracted using hot MeOH. The concentrated MeOH extract (115 g) was fractionated using a Diaion HP-20 porous polymer polystyrene resin column with MeOH/H₂O (3 : 7), MeOH/H₂O (1 : 1), MeOH, EtOH, and EtOAc, successively (each solvent volume 6 L). The MeOH eluted fraction was subjected to silica gel column chromatography (CC), octadecylsilanized (ODS) silica gel CC, and preparative ODS HPLC to obtain 28 compounds (1–28). Previously characterized compounds (22–28) were identified as (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (22),¹⁹⁾ (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (23),¹⁹⁾ (23S,24S)-3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (24),²⁰⁾ (23S,24S)-21-acetyloxy-3β,23,24-trihydroxyspirosta-5,25(27)-dien-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside (25),¹⁰⁾ (25R)-26-[(β-D-glucopyranosyl)oxy]-1β,22α-dihydroxyfurost-5-en-3β-yl β-D-glucopyranoside (26),²¹⁾ (25R)-26-[(β-D-glucopyranosyl)oxy]-22α-hydroxyfurost-5-en-3β-yl O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (27),²²⁾ and (25S)-22α,25-epoxy-26-[(β-D-glucopyranosyl)oxy]-3β-hydroxyfurost-5-en-1β-yl α-L-arabinopyranoside (28),¹³⁾ respectively (Fig. 1).

Fig. 1. Structures of Isolated Compounds 1–28

Ara: α-L-arabinopyranosyl; Api: β-D-apiofuranosyl; Fuc: β-D-fucopyranosyl; Gal: β-D-galactopyranosyl; Glc: β-D-glucopyranosyl; Rha: α-L-rhamnopyranosyl; Qui: β-D-quinovopyranosyl; Ac: acetyl.

Compound 1 was obtained as an amorphous powder and its molecular formula was determined to be C₄₅H₇₀O₁₉, based on high resolution-electrospray ionization-time of flight (HR-ESI-TOF)-MS and ¹³C-NMR spectra data. The IR spectrum of 1 showed absorption bands of hydroxy (3392 cm⁻¹) and carbonyl groups (1729 cm⁻¹). The ¹H- and ¹³C-NMR spectra of 1 indicated the presence of four steroidal methyl groups [δ_H 1.35 (3H, s, Me-19), 1.13 (3H, d, J = 7.0 Hz, Me-21), 1.02 (3H, s, Me-18), and 0.97 (3H, d, J = 6.8 Hz, Me-27); δ_C 16.8 (C-18), 14.9 (C-19), 14.6 (C-21), and 13.0 (C-27)], an olefinic group [δ_H 5.60 (1H, br d, J = 5.8 Hz, H-6); δ_C 139.3 (C-5) and 124.9 (C-6)], an oxygenated methylene group [δ_H 3.82 (1H, dd, J = 11.2, 11.2 Hz, H-26ax) and 3.43 (1H, dd, J = 11.2, 4.7 Hz, H-26eq); δ_C 60.6 (C-26)], five oxygenated methine groups [δ_H 4.61 (1H, m, H-16), 4.10 (1H, br s, H-24), 3.90 (1H, m, H-3), 3.83 (1H, dd, J = 12.0, 4.1 Hz, H-1), and 3.81 (1H, d, J = 3.2 Hz, H-23); δ_C 84.0 (C-1), 83.0 (C-16), 73.0 (C-24), 68.7 (C-23), and 68.0 (C-3)], and an acetyl group [δ_H 2.01 (3H, s); δ_C 170.8 and 21.0]. Furthermore, signals corresponding to three anomeric protons and carbons were observed [δ_H 6.48 (1H, br s), 4.94 (1H, d, J = 7.6 Hz), and 4.69 (1H, d, J = 7.9 Hz); δ_C 106.7, 100.8, and 100.7]. The ¹H- and ¹³C-NMR spectral data of 1 were similar to those obtained for 24, except for the signals arising from the F-ring part of the steroidal skeleton. Instead of the ¹H- and ¹³C-NMR signals arising from the C-25(27) exomethylene group [δ_H 5.07 and 4.97 (each 1H, br s, H₂-27); δ_C 146.1 (C-25) and 112.3 (C-27)] observed in 24, signals from a secondary methyl group [δ_H 0.97 (3H, d, J = 6.8 Hz, Me-27); δ_C 13.0 (C-27)] and methine group [δ_H 1.89 (1H, m, H-25); δ_C 36.1 (C-25)] were observed in the ¹H- and ¹³C-NMR spectra of 1. These data suggest that 1 is a saturated derivative of 24 in terms of the C-25(27) exomethylene group. Nuclear Overhauser effect spectroscopy (NOESY) of 1 was performed and NOE correlations were observed between H-23 (δ_H 3.81) and H-20 (δ_H 2.97)/H-24 (δ_H 4.10)/H-25 (δ_H 1.89) as well as between H-26ax (δ_H 3.82) and H-16 (δ_H 4.61). Additionally, the proton spin-coupling constants of ³J_H-23,H-24 and ³J_H-25,H-26ax were 3.2 and 11.2 Hz, respectively. Thus, the configurations of C-23, C-24, and C-25 were determined as 23S, 24S, and 25S. Acid hydrolysis of 1 with 0.2 M HCl (dioxane/H₂O; 1 : 1) yielded L-arabinose, L-rhamnose, and D-xylose carbohydrate moieties. The ¹H-¹H correlation spectroscopy (COSY) and heteronuclear single quantum coherence (HSQC) spectra indicated that the sugar moiety of 1 comprised a 2,3-disubstituted inner α-L-arabinopyranosyl unit [Ara: δ_H 4.69 (1H, d, J = 7.9 Hz, H-1′); δ_C 100.8, 72.8, 85.2, 69.9, and 67.3 (C-1′–5′)], a terminal α-L-rhamnopyranosyl unit [Rha: δ_H 6.48 (1H, br s, H-1″); δ_C 100.7, 72.2, 69.9, 76.5, 66.5, and 18.5 (C-1″–6″)], and a terminal β-D-xylopyranosyl unit [Xyl: δ_H 4.94 (1H, d, J = 7.6 Hz, H-1‴); δ_C 106.7, 74.5, 78.5, 70.9, and 67.1 (C-1‴–5‴)]. In the heteronuclear multiple bond correlation (HMBC) spectrum of 1, long-range correlations were observed between H-1″ of Rha (δ_H 6.48) and C-2′ of Ara (δ_C 72.8), H-1‴ of Xyl (δ_H 4.94) and C-3′ of Ara (δ_C 85.2), between H-1′ of Ara (δ_H 4.69) and C-1 of the aglycone (δ_C 84.0). Furthermore, a ³J_C,H correlation was observed between H-4″ of Rha (δ_H 5.78) and the acetyl carbonyl carbon (δ_C 170.8). Accordingly, 1 was determined to be (23S,24S,25S)-3β,23,24-trihydroxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 2 (C₅₁H₈₀O₂₃) indicated that 2 was analogous to 1, including the triglycoside moiety attached to C-1 of the aglycone. However, the molecular formula of 2 was larger than that of 1 by a C₆H₁₀O₄ unit, indicating the presence of an additional hexosyl unit. Acid hydrolysis of 2 yielded L-arabinose, L-rhamnose, D-quinovose, and D-xylose, while ¹H-¹H COSY and HSQC analysis indicated that 2 contained a terminal β-D-quinovopyranosyl unit [Qui: δ_H 5.24 (1H, d, J = 7.6 Hz, H-1″″); δ_C 105.3, 76.2, 78.1, 76.9, 73.0, and 18.6 (C-1″″–6″″)]. Comparing the ¹³C-NMR spectrum of 2 with that of 1, the signal attributed to C-24 of the aglycone was shifted downfield by 8.4 ppm. Additionally, the HMBC spectrum of 2 showed a long-range correlation between H-1″″ of Qui (δ_H 5.24) and C-24 of the aglycone (δ_C 81.4). Therefore, 2 was determined to be (23S,24S,25S)-24-[(β-D-quinovopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Comparison of the ¹H- and ¹³C-NMR spectra of 3 (C₅₀H₇₈O₂₃) with those of 1 suggested that 3 contained the same aglycone moiety as 1 but differed in terms of the sugar moiety attached to C-1 of the aglycone. Acid hydrolysis of 3 afforded L-arabinose, D-apiose, L-rhamnose, and D-xylose, while ¹H-¹H COSY and HSQC analysis of the sugar moiety in 3 showed that it consisted of a 2,3-disubstituted α-L-arabinopyranosyl (Ara), 3,4-disubstituted α-L-rhamnopyranosyl (Rha), terminal β-D-xylopyranosyl (Xyl), and terminal β-D-apiofuranosyl units [Api: δ_H 5.95 (1H, br s, H-1″″); δ_C 112.1, 77.8, 80.0, 74.8, and 65.2 (C-1″″–5″″)], as well as an acetyl group. The α-anomeric configuration of Rha was determined by the large ¹J_C-1,H-1 value of 169.7 Hz. In the HMBC spectrum of 3, long-range correlations were observed between H-1″″ of Api (δ_H 5.95) and C-3″ of Rha (δ_C 77.7), H-1″ of Rha (δ_H 6.49) and C-2′ of Ara (δ_C 72.4), H-1‴ of Xyl (δ_H 4.92) and C-3′ of Ara (δ_C 85.2), H-4″ of Rha (δ_H 5.90) and the acetyl carbonyl carbon (δ_C 170.7), and between H-1′ of Ara (δ_H 4.63) and C-1 of the aglycone (δ_C 84.2). Thus, 3 was determined to be (23S,24S,25S)-3β,23,24-trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Compound 4 (C₅₆H₈₈O₂₇) exhibited ¹H- and ¹³C-NMR spectral features similar to those of 3, except for the signals arising from the F-ring part of the steroidal skeleton. The molecular formula of 4 featured an additional C₆H₁₀O₄ compared to that of 3. From acid hydrolysis of 4, L-arabinose, D-apiose, D-fucose, L-rhamnose, and D-xylose were obtained. A terminal β-D-fucopyranosyl unit [Fuc: δ_H 5.13 (1H, d, J = 7.9 Hz, H-1″‴); δ_C 105.8, 73.1, 75.1, 72.6, 71.3, and 17.1 (C-1″‴–6″‴)] was detected by ¹H-¹H COSY and HSQC spectral analysis of 4. Comparing the ¹³C-NMR spectrum of 4 with that of 3, the signal assigned to C-24 of the aglycone was shifted downfield by 8.4 ppm and a ³J_C,H correlation was observed between H-1″‴ of Fuc (δ_H 5.13) and C-24 of the aglycone (δ_C 81.4) in the HMBC spectrum. Therefore, 4 was elucidated as (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 5 (C₄₉H₇₈O₂₃) were essentially analogous to those of 22, but remarkable differences were observed for the signals assigned to the monosaccharide linked to C-24 of the aglycone between the two glycosides. Acid hydrolysis of 5 yielded L-arabinose, D-glucose, L-rhamnose, and D-xylose. Analysis of the associated ¹H-¹H COSY and HSQC spectra indicated the presence of a terminal β-D-glucopyranosyl unit [Glc: δ_H 5.33 (1H, d, J = 7.7 Hz, H-1″″); δ_C 105.6, 76.1, 78.3, 69.5, 78.3, and 63.0 (C-1″″–6″″)]. The HMBC spectrum of 5 showed a long-range correlation between H-1″″ of Glc (δ_H 5.33) and C-24 of the aglycone (δ_C 81.6). Accordingly, 5 was determined to be (23S,24S,25S)-24-[(β-D-glucopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 6 (C₄₅H₇₀O₂₀), 7 (C₅₁H₈₀O₂₄), and 8 (C₅₀H₇₈O₂₄) were closely related to those of 1, 23, and 3, respectively. The molecular formula of 6 was larger than that of 1 (C₄₅H₇₀O₁₉) by a single oxygen atom, and only differences observed in the ¹H- and ¹³C-NMR signals were those assigned to Me-21/C-21. The methyl signals [δ_H 1.13 (3H, d, J = 7.0 Hz); δ_C 14.6] in 1 were displaced by hydroxymethyl signals [δ_H 4.22 (1H, br d, J = 10.1 Hz) and 4.01 (1H, dd, J = 10.1, 6.7 Hz); δ_C 62.3] in 6. Similarly, the molecular formulas of 7 and 8 were larger than those of 23 (C₅₁H₈₀O₂₃) and 3 (C₅₀H₇₈O₂₃) by a single oxygen atom, and the C-21 methyl groups were hydroxylated, as indicated by the ¹H- and ¹³C-NMR spectra [7: δ_H 4.20 (1H, br d, J = 10.4 Hz) and 4.01 (1H, dd, J = 10.4, 7.1 Hz); δ_C 62.5; 8: δ_H 4.19 (1H, m), 3.98 (1H, m); δ_C 62.3)]. Therefore, 6, 7, and 8 were identified as the corresponding C-21 hydroxy derivatives of 1, 23, and 3, respectively, establishing them as (23S,24S,25S)-3β,21,23,24-tetrahydroxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,21,23-trihydroxyspirost-5-en-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, and (23S,24S,25S)-3β,21,23,24-tetrahydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, respectively.

The ¹H- and ¹³C-NMR spectra of 9 (C₅₅H₈₆O₂₈) were similar to those obtained for 8, except for the presence of the signals attributed to one more monosaccharide unit. The molecular formula of 9 was larger than that of 8 by C₅H₈O₄, corresponding to a single pentosyl unit. Acid hydrolysis of 9 yielded L-arabinose, D-apiose, L-rhamnose, and D-xylose. Analysis of the corresponding ¹H-¹H COSY and HSQC spectra of the sugar moieties of 9 indicated the presence of an additional terminal α-L-arabinopyranosyl unit [Ara′: δ_H 5.11 (1H, d, J = 7.7 Hz, H-1″‴); δ_C 106.4, 73.6, 74.8, 69.9, and 67.2 (C-1″‴–5″‴)]. A long-range correlation was observed between H-1″‴ of Ara′ (δ_H 5.11) and C-24 of the aglycone (δ_C 81.7) in the HMBC spectrum. Thus, 9 was identified as (23S,24S,25S)-24-[(β-D-arabinopyranosyl)oxy]-3β,21,23-trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Analysis of the ¹H- and ¹³C-NMR spectra of 10 (C₅₆H₈₈O₂₈) and subsequent comparison with those of 9 revealed that the aglycone and tetraglycoside attached to C-1 of the aglycone were identical to those of 9 but differed in terms of the monosaccharide linked to C-24 of the aglycone. Instead of the signals for an arabinopyranosyl moiety, signals attributed to a β-D-fucopyranosyl (Fuc) residue were observed. Acid hydrolysis of 10 yielded L-arabinose, D-apiose, D-fucose, L-rhamnose, and D-xylose. The linkage of the Fuc moiety to C-24 of the aglycone was confirmed by an HMBC correlation between H-1″‴ of Fuc (δ_H 5.13) and C-24 of the aglycone (δ_C 81.7). Thus, 10 was assigned as (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-3β,21,23-trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 11 (C₅₆H₈₈O₂₉) suggested that it was analogous to 8, including the tetraglycoside moiety attached to C-1 of the aglycone. The molecular formula of 11 was larger than that of 8 by C₆H₁₀O₅, corresponding to a single hexosyl unit. Acid hydrolysis of 11 yielded L-arabinose, D-apiose, D-galactose, L-rhamnose, and D-xylose. The ¹H-¹H COSY and HSQC spectral analysis of 11 indicated the presence of a terminal β-D-galactopyranosyl unit [Gal: δ_H 4.90 (1H, d, J = 7.9 Hz, H-1″‴); δ_C 105.4, 72.5, 75.6, 70.2, 77.0, and 62.4 (C-1″‴–6″‴)], and an HMBC correlation was observed between H-1″‴ of Gal (δ_H 4.90) and C-21 of the aglycone (δ_C 70.0). Therefore, 11 was determined to be (23S,24S,25S)-21-[(β-D-galactopyranosyl)oxy]-3β,23,24-trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 12 (C₆₁H₉₆O₃₃) were similar to those obtained for 11, but the molecular formula of 12 was larger than that of 11 by C₅H₈O₄. Acid hydrolysis of 12 afforded L-arabinose, D-apiose, D-galactose, L-rhamnose, and D-xylose. The ¹H-¹H COSY and HSQC spectral analysis of 12 indicated the presence of signals corresponding to an additional terminal α-L-arabinopyranosyl (Ara′) unit. The HMBC spectrum of 12 contained a long-range correlation from H-1‴‴ of Ara′ (δ_H 5.10) to C-24 of the aglycone (δ_C 81.4). Thus, 12 was assigned as (23S,24S,25S)-24-[(β-D-arabinopyranosyl)oxy]-21-[(β-D-galactopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Compound 13 (C₆₁H₉₈O₃₃) was obtained as an amorphous powder and its ¹H- and ¹³C-NMR spectra showed the same aglycone unit as 7, also indicating that the sugar moieties attached to C-1 and C-24 of the aglycone of 13 were coincident with those of 22. Additionally, a 2-substituted β-D-galactopyranosyl unit [Gal: δ_H 4.98 (1H, d, J = 7.4 Hz, H-1″″); δ_C 103.4, 81.6, 75.2, 69.7, 77.0, and 62.2 (C-1″″–6″″)] and a terminal β-D-glucopyranosyl unit [Glc: δ_H 5.40 (1H, d, J = 7.6 Hz, H-1″‴); δ_C 106.1, 76.5, 78.1, 71.4, 78.6, and 62.5 (C-1″‴–6″‴)] were detected in the ¹H-¹H COSY and HSQC spectral analysis of 13. Acid hydrolysis of 13 afforded L-arabinose, D-fucose, D-galactose, D-glucose, L-rhamnose, and D-xylose. In the HMBC spectrum of 13, long-range correlations were observed between H-1″‴ of Glc (δ_H 5.40) and C-2″″ of Gal (δ_C 81.6) as well as between H-1″″ of Gal (δ_H 4.98) and C-21 of the aglycone (δ_C 69.9). Thus, 13 was identified as (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-21-[O-β-D-glucopyranosyl-(1→2)-(β-D-galactopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-α-L-rhamnopyranosyl-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

The ¹H- and ¹³C-NMR spectra of 14 (C₆₂H₉₈O₃₄) and 15 (C₆₈H₁₀₈O₃₈) indicated their relation to 8 and 10, respectively, containing a diglycoside composed of Glc and Gal at C-21 of the aglycone, as in 13. The molecular formula of 14 and 15 were both larger than those of 8 and 10, respectively, by C₁₂H₂₀O₁₀ and acid hydrolysis of 14 yielded L-arabinose, D-galactose, D-glucose, L-rhamnose, and D-xylose. In contrast, acid hydrolysis of 15 afforded L-arabinose, D-fucose, D-galactose, D-glucose, L-rhamnose, and D-xylose. In the HMBC spectra of 14 and 15, long-range correlations were observed between H-1‴‴ of Glc [δ_H 5.38 (14); 5.38 (15)] and C-2″‴ of Gal [δ_C 81.6 (14); 81.8 (15)] as well as between H-1″‴ of Gal [δ_H 4.98 (14); 4.97 (15)] and C-21 of the aglycone [δ_C 69.8 (14); 69.8 (15)]. Accordingly, 14 and 15 were identified as (23S,24S,25S)-21-[O-(β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl)oxy]-3β,23,24-trihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside and (23S,24S,25S)-24-[(β-D-fucopyranosyl)oxy]-21-[O-(β-D-glucopyranosyl-(1→2)-β-D-galactopyranosyl)oxy]-3β,23-dihydroxyspirost-5-en-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, respectively.

The NMR spectral features of 16 (C₅₆H₈₆O₂₇), 17 (C₅₀H₇₆O₂₄), and 18 (C₅₅H₈₄O₂₈) were related to those of 4, 8, and 9, respectively. However, instead of containing three-proton doublet signals for Me-27, as observed in the ¹H- and ¹³C-NMR spectra of 4, 8, and 9, signals arising from an exomethylene group [δ_H 5.23 (br s) and 5.08 (br s) (16); 5.05 (br s) and 4.95 (br s) (17); 5.20 (br s) and 5.06 (br s) (18); δ_C 113.7 (16); 112.3 (17); 113.9 (18)] and quaternary carbon [δ_C 143.8 (16); 146.2 (17); 143.7 (18)] were observed. Therefore, 16, 17, and 18 correspond to the C-25/27 dehydroxy derivatives of 4, 8, and 9, respectively, as additionally confirmed by long-range correlations from H₂-27 to C-24, C-25, and C-26 in the associated HMBC spectra. Therefore, 16, 17, and 18 were identified as (23S,24S)-24-[(β-D-fucopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-dien-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, (23S,24S)-3β,21,23,24-tetrahydroxyspirosta-5,25(27)-dien-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, and (23S,24S)-24-[(α-L-arabinopyranosyl)oxy]-3β,21,23-trihydroxyspirosta-5,25(27)-dien-1β-yl O-β-D-apiofuranosyl-(1→3)-O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside, respectively.

Comparison of the ¹H- and ¹³C-NMR spectra 19 (C₅₇H₈₆O₂₉) with those of 18 showed considerable structural similarity. Furthermore, the presence of an additional acetyl group to that attached at C-21 of the aglycone was indicated by the ¹H- and ¹³C-NMR spectra [δ_H 1.92 (3H, s); δ_C 170.8 and 20.9]. The ester linkage at aglycone C-21 was formed from acetic acid, as indicated by the long-range correlations between the H₂-21 methylene protons [δ_H 4.37 (m) and 4.34 (m)] and acetyl carbonyl carbon (δ_C 170.8). Thus, 19 was identified as (23S,24S)-21-acetyloxy-24-[(α-L-arabinopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-dien-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Analysis of the ¹H- and ¹³C-NMR spectra of 20 (C₅₂H₇₈O₂₅) and comparison with those of 19 showed that the aglycone with an acetyloxy group at C-21 and monosaccharide at C-24 were identical to those observed in 19. However, the ¹H- and ¹³C-NMR signals for the apiofuranosyl group linked to C-3 of the rhamnopyranosyl unit in the sugar moiety attached to the aglycone C-1 were not observed. The ¹H- and ¹³C-NMR spectra of the triglycoside moiety agreed with those observed in the concomitantly isolated spirostanol triglycosides 1, 2, 6, and 7. Thus, 20 was identified as (23S,24S)-21-acetyloxy-24-[(α-L-arabinopyranosyl)oxy]-3β,23-dihydroxyspirosta-5,25(27)-dien-1β-yl O-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→2)-O-[β-D-xylopyranosyl-(1→3)]-α-L-arabinopyranoside.

Compound 21 was obtained as an amorphous powder with a molecular formula was of C₃₈H₆₂O₁₄ as determined by HR-ESI-TOF-MS and ¹³C-NMR data. In the ¹H- and ¹³C-NMR spectra of 21, signals corresponding to four steroidal methyl groups [δ_H 1.27 (3H, d, J = 7.1 Hz, Me-21), 1.21 (3H, s, Me-19), 0.95 (3H, d, J = 6.5 Hz, Me-27), and 0.90 (3H, s, M-18); δ_C 17.4 (C-27), 16.7 (C-18), 16.3 (C-21), and 14.7 (C-19)], an olefinic group [δ_H 5.55 (1H, br d, J = 4.6 Hz, H-6); δ_C 139.5 (C-5) and 124.7 (C-6)], two anomeric protons and associated carbons [δ_H 4.79 (1H, d, J = 7.7 Hz) and 4.75 (1H, d, J = 7.1 Hz); δ_C 104.8 and 102.2], and a hemiacetal carbon [δ_C 110.6 (C-22)] were observed. These data suggested that 21 was a 22-hydroxyfurostanol glycoside and its enzymatic hydrolysis with naringinase yielded (25R)-3β-hydroxyspirost-5-en-1β-yl α-L-arabinopyranoside (21a)²²⁾ and D-glucose. In the associated HMBC spectrum, a long-range correlation was observed between H-1″ of the β-D-glucopyranosyl unit (δ_H 4.79) and C-26 of the aglycone (δ_C 75.2). Accordingly, 21 was identified as (25R)-26-[(β-D-glucopyranosyl)oxy]-3β,22α-dihydroxyfurost-5-en-1β-yl α-L-arabinopyranoside.

Compounds 12, 13, and 15 are spirostanol trisdesmosides, which are types of steroidal glycosides rarely observed in the plant kingdom.

The cytotoxic activities of 1–28 were evaluated against HL-60 and A549 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Compound 7 showed cytotoxic activity against HL-60 and A549 cells with IC₅₀ values of 5.9 ± 0.46 and 6.6 ± 0.17 µM, respectively. In contrast, 19 was selectively cytotoxic against only A549 cells with an IC₅₀ value of 5.5 ± 0.61 µM. Cisplatin was used as a positive control, yielding IC₅₀ values of 1.9 and 3.7 µM for HL-60 and A549 cells, respectively. Compounds 1–6, 8–18, and 20–28 did not show cytotoxicity against HL-60 and A549 cells at concentrations of up to 10 µM.

Experimental

General

Optical rotations were measured on a JASCO P-1030 automatic digital polarimeter (Jasco, Tokyo, Japan). IR spectra were obtained using a JASCO FT-IR 620 spectrophotometer (Jasco). NMR spectra were recorded on a Bruker DRX-500 or AV-600 spectrometer (Bruker, Karlsruhe, Germany) using standard Bruker pulse programs. Chemical shifts (δ) were given with reference to tetramethylsilane (TMS) as an internal standard. HR-ESI-TOF-MS data were obtained using a Water-Micromass LCT mass spectrometer (Waters-Micromass, Manchester, U.K.). CC was conducted by Diaion HP-20 (Mitsubishi-chemical, Tokyo, Japan), silica gel Chromatorex BW-300 (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel COSMOSIL 75C₁₈-OPN (Nacalai Tesque, Kyoto, Japan). TLC was carried out precoated silica gel 60 F₂₅₄ or RP₁₈ F₂₅₄S plates (0.25 mm thick) (Merck, Darmstadt, Germany), and the spots were detected by spraying the plates with 10% H₂SO₄ aqueous solution and then heating. HPLC was conducted using a system consisting of a CCPM pump (Shimadzu, Kyoto, Japan), an RI-8021 (Tosoh, Tokyo, Japan) or a Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector, and a Rheodyne injection port (Rohnert Park, CA, U.S.A.). A TSK gel ODS-100Z column (10 mm i.d. × 250 mm, 5 µm) (Tosoh) was used for the preparative HPLC. Purities of the isolated compounds were verified by MS, TLC, and NMR spectra. The following materials and biochemical grade reagents were used for the cell cultures and cytotoxicity assays: SH-1300 Lab microplate reader (CORONA ELECTRIC, Ibaraki, Japan); 96-well flat-bottom plate (Iwaki Glass, Chiba, Japan); fetal bovine serum (FBS), 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution, RPMI-1640 medium, minimum essential medium (MEM), cisplatin, and MTT (Sigma, St. Louis, MO, U.S.A.); paraformaldehyde and phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan); penicillin G sodium salt and streptomycin sulfate (Gibco, Gland Island, NY, U.S.A.); HL-60 cells (JCRB0085) and A549 cells (JCRB0076) (Human Science Research Resources Bank, Osaka, Japan).

Plant Material

The whole plants of H. foetidus were purchased from Fuji-engei (Okayama, Japan). A voucher specimen was deposited at the herbarium of the Tokyo University of Pharmacy and Life Sciences (KS-2014-011).²³⁾

Extraction and Isolation

The whole plants of H. foetidus (fresh weight, 3.3 kg) were extracted with hot MeOH (10 L). After solvent was removed in vacuo, the concentrated MeOH extract (115 g) was fractionated by Diaion HP-20 column and then successively eluted with MeOH/H₂O (3 : 7), MeOH/H₂O (1 : 1), MeOH, EtOH, and EtOAc (each solvent volume 6 L). The MeOH eluate fraction (10 g) was chromatographed on silica gel eluted with stepwise gradient of CHCl₃/MeOH/H₂O (90 : 10 : 1, 40 : 10 : 1, 20 : 10 : 1) and finally MeOH alone to get 17 fractions (Frs. 1–17). Fraction 10 was subjected to silica gel CC eluted with CHCl₃/MeOH/H₂O (90 : 10 : 1; 40 : 10 : 1) and EtOAc/MeOH/H₂O (80 : 10 : 1; 50 : 10 : 1; 40 : 10 : 1), and ODS silica gel CC eluted with MeOH/H₂O (3 : 2; 1 : 1) and MeCN/H₂O (7 : 3; 1 : 1; 3 : 7; 1 : 3) to yield 1 (35.7 mg), 21 (85.4 mg), 23 (72.1 mg), and 24 (2.9 mg). Fraction 11 was separated by ODS silica gel CC using MeOH/H₂O (4 : 1; 3 : 2) and MeCN/H₂O (1 : 3) to obtain 2 (5.8 mg), 3 (27.0 mg), 6 (3.3 mg), 25 (18.1 mg), and 28 (6.7 mg). Fraction 13 was chromatographed on silica gel eluted with CHCl₃/MeOH/H₂O (30 : 10 : 1), ODS silica gel eluted with MeOH/H₂O (3 : 2) and MeCN/H₂O (1 : 3; 1 : 4), and preparative HPLC using MeCN/H₂O (1 : 2; 3 : 7) to furnish 4 (72.1 mg), 5 (2.3 mg), 7 (30.1 mg), 8 (13.0 mg), 20 (8.5 mg), and 26 (85.4 mg). Fraction 14 was fractionated by ODS silica gel CC using MeOH/H₂O (3 : 2; 11 : 9; 1 : 1) and MeCN/H₂O (1 : 3; 1 : 4), and preparative HPLC using MeCN/H₂O (3 : 7; 1 : 3) to acquire 16 (16.2 mg), 17 (6.1 mg), 19 (28.6 mg), 22 (16.6 mg), and 27 (4.9 mg). Fraction 15 was separated by ODS silica gel CC eluted with MeOH/H₂O (11 : 9; 1 : 1) and MeCN/H₂O (1 : 3; 1 : 4), and preparative HPLC using MeCN/H₂O (1 : 3) to afford 9 (26.9 mg) and 10 (6.8 mg). Fraction 16 was chromatographed on ODS silica gel using MeOH/H₂O (11 : 9; 9 : 11) and MeCN/H₂O (1 : 3; 1 : 4) to collect 11 (3.9 mg) and 18 (46.3 mg). Fraction 17 was subjected to silica gel CC eluted with CHCl₃/MeOH/H₂O (20 : 10 : 1; 10 : 10 : 1), ODS silica gel CC eluted with MeOH/H₂O (1 : 1) and MeCN/H₂O (1 : 3; 1 : 4), and preparative HPLC using MeCN/H₂O (1 : 3; 1 : 4) to furnish 12 (12.9 mg), 13 (7.0 mg), 14 (19.9 mg), and 15 (15.9 mg).

Compound 1

Amorphous powder; [α]_D²⁵ −94.4 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3392 (OH), 2926 (CH), 1729 (C=O); ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 937.4412 [M + Na]⁺ (Calcd for C₄₅H₇₀NaO₁₉: 937.4409).

Table 1. ¹H-NMR Data for the Aglycone Moiety of 1–21

All spectra were measured in C₅D₅N (1, 8, 18, and 19: 500 MHz; 2-7, 9-17, 20, and 21: 600 MHz). 1) Signals are unclear due to overlapping with water signal.

Table 2. ¹³C-NMR Data for the Aglycone Moiety of 1–21

All spectra were measured in C₅D₅N (1, 8, 18, and 19: 125 MHz; 2–7, 9–17, 20, and 21: 150 MHz).

Table 3. ¹H-NMR Data for the Sugar and Acetyl Moieties of 1–21

All spectra were measured in C₅D₅N (1, 8, 18, and 19: 500 MHz; 2–7, 9–17, 20, and 21: 600 MHz).

Table 4. ¹³C-NMR Data for the Sugar and Acetyl Moieties of 1–21

All spectra were measured in C₅D₅N (1, 8, 18, and 19: 125 MHz; 2–7, 9–17, 20, and 21: 150 MHz).

Compound 2

Amorphous powder; [α]_D²⁵ −14.5 (c = 0.05, MeOH); IR ν_max (film) cm⁻¹: 3370 (OH), 2925 (CH), 1730 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1083.4980 [M + Na]⁺ (Calcd for C₅₁H₈₀NaO₂₃: 1083.4988).

Compound 3

Amorphous powder; [α]_D²⁵ −23.4 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3375 (OH), 2930 (CH), 1727 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1069.4832 [M + Na]⁺ (Calcd for C₅₀H₇₈NaO₂₃: 1069.4832).

Compound 4

Amorphous powder; [α]_D²⁵ −31.7 (c = 0.11, MeOH); IR ν_max (film) cm⁻¹: 3375 (OH), 2927 (CH), 1727 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1215.5405 [M + Na]⁺ (Calcd for C₅₆H₈₈NaO₂₇: 1215.5411).

Compound 5

Amorphous powder; [α]_D²⁵ −8.7 (c = 0.14, MeOH); IR ν_max (film) cm⁻¹: 3374 (OH), 2925 (CH); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1057.4819 [M + Na]⁺ (Calcd for C₄₉H₇₈NaO₂₃: 1057.4832).

Compound 6

Amorphous powder; [α]_D²⁵ −30.5 (c = 0.05, MeOH); IR ν_max (film) cm⁻¹: 3364 (OH), 2925 (CH), 1725 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 953.4354 [M + Na]⁺ (Calcd for C₄₅H₇₀NaO₂₀: 953.4358).

Compound 7

Amorphous powder; [α]_D²⁵ −42.0 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3383 (OH), 2922 (CH), 1728 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1099.4934 [M + Na]⁺ (Calcd for C₅₁H₈₀NaO₂₄: 1099.4937).

Compound 8

Amorphous powder; [α]_D²⁵ −13.9 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3389 (OH), 2924 (CH), 1729 (C=O); ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1085.4775 [M + Na]⁺ (Calcd for C₅₀H₇₈NaO₂₄: 1085.4781).

Compound 9

Amorphous powder; [α]_D²⁵ −62.5 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3388 (OH), 2925 (CH), 1731 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1217.5203 [M + Na]⁺ (Calcd for C₅₅H₈₆NaO₂₈: 1217.5203).

Compound 10

Amorphous powder; [α]_D²⁵ −41.9 (c = 0.05, MeOH); IR ν_max (film) cm⁻¹: 3389 (OH), 2925 (CH), 1731 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1231.5364 [M + Na]⁺ (Calcd for C₅₆H₈₈NaO₂₈: 1231.5360).

Compound 11

Amorphous powder; [α]_D²⁵ −8.8 (c = 0.05, MeOH); IR ν_max (film) cm⁻¹: 3357 (OH), 2926 (CH), 1731 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1247.5299 [M + Na]⁺ (Calcd for C₅₆H₈₈NaO₂₉: 1247.5309).

Compound 12

Amorphous powder; [α]_D²⁵ −22.7 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3357 (OH), 2930 (CH), 1732 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1379.5753 [M + Na]⁺ (Calcd for C₆₁H₉₆NaO₃₃: 1379.5732).

Compound 13

Amorphous powder; [α]_D²⁵ −17.7 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3364 (OH), 2927 (CH); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1381.5883 [M + Na]⁺ (Calcd for C₆₁H₉₈NaO₃₃: 1381.5888).

Compound 14

Amorphous powder; [α]_D²⁵ −34.1 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3360 (OH), 2925 (CH), 1730 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1409.5863 [M + Na]⁺ (Calcd for C₆₂H₉₈NaO₃₄: 1409.5837).

Compound 15

Amorphous powder; [α]_D²⁵ −32.0 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3373 (OH), 2931 (CH), 1731 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1555.6422 [M + Na]⁺ (Calcd for C₆₈H₁₀₈NaO₃₈: 1555.6416).

Compound 16

Amorphous powder; [α]_D²⁵ −69.1 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3389 (OH), 2932 (CH), 1732 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1213.5251 [M + Na]⁺ (Calcd for C₅₆H₈₆NaO₂₇: 1213.5254).

Compound 17

Amorphous powder; [α]_D²⁵ −94.4 (c = 0.05, MeOH); IR ν_max (film) cm⁻¹: 3375 (OH), 2925 (CH), 1731 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1083.4623 [M + Na]⁺ (Calcd for C₅₀H₇₆NaO₂₄: 1083.4624).

Compound 18

Amorphous powder; [α]_D²⁵ −125.4 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3404 (OH), 2923 (CH), 1728 (C=O); ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1215.5081 [M + Na]⁺ (Calcd for C₅₅H₈₄NaO₂₈: 1215.5047).

Compound 19

Amorphous powder; [α]_D²⁵ −110.3 (c = 0.10, MeOH); IR ν_max (film) cm⁻¹: 3393 (OH), 2924 (CH), 1733 (C=O); ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (500 MHz, C₅D₅N) and ¹³C-NMR (125 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1257.5111 [M + Na]⁺ (Calcd for C₅₇H₈₆NaO₂₉: 1257.5152).

Compound 20

Amorphous powder; [α]_D²⁵ −43.6 (c = 0.025, MeOH); IR ν_max (film) cm⁻¹: 3406 (OH), 2923 (CH), 1720 (C=O); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar and acetyl moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 1125.4730 [M + Na]⁺ (Calcd for C₅₂H₇₈NaO₂₅: 1125.4730).

Compound 21

Amorphous powder; [α]_D²⁵ −10.7 (c = 0.11, MeOH); IR ν_max (film) cm⁻¹: 3364 (OH), 2925 (CH); ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the aglycone moiety, see Tables 1, 2; ¹H- (600 MHz, C₅D₅N) and ¹³C-NMR (150 MHz, C₅D₅N) data of the sugar moieties, see Tables 3, 4; HR-ESI-TOF-MS m/z: 765.4035 [M + Na]⁺ (Calcd for C₃₈H₆₂NaO₁₄: 765.4037).

Acid Hydrolysis of 1–20

Compound 1 (10.0 mg), 2 (2.0 mg), 3 (3.6 mg), 4 (4.0 mg), 5 (1.9 mg), 6 (1.1 mg), 7 (10.0 mg), 8 (5.0 mg), 9 (5.5 mg), 10 (2.3 mg), 11 (2.5 mg), 12 (4.7 mg), 13 (4.0 mg), 14 (5.0 mg), 15 (5.0 mg), 16 (4.8 mg), 17 (2.4 mg), 18 (10.0 mg), 19 (5.0 mg), and 20 (2.8 mg) were independently dissolved in 0.2 M HCl or 0.5 M HCl (dioxane/H₂O, 1 : 1) and heated at 95°C for 30 min under an Ar atmosphere. Each reaction mixture was neutralized by passing through an Amberlite IRA-96 (Organo, Tokyo, Japan) column, was subjected to Diaion HP-20 CC eluted with MeOH/H₂O (2 : 3) and finally Me₂CO/EtOH (1 : 1) to obtain sugar fractions (3.3 mg from 1; 0.79 mg from 2; 1.4 mg from 3; 2.9 mg from 4; 0.77 mg from 5; 0.38 mg from 6; 4.5 mg from 7; 2.8 mg from 8; 2.3 mg from 9; 0.92 mg from 10; 0.8 mg from 11; 2.3 mg from 12; 2.0 mg from 13; 2.5 mg from 14; 2.6 mg from 15; 2.1 mg from 16; 0.94 mg from 17; 3.8 mg from 18; 1.9 mg from 19; and 1.0 mg from 20). The sugar fractions were analyzed by HPLC under the following conditions: column, Capcell Pak NH₂ UG80 (4.6 mm i.d.  × 250 mm, 5 µm, Shiseido, Tokyo, Japan); solvent, MeCN/H₂O (17 : 3); detection, refractive index and optical rotation; flow rate, 1.0 mL/min. D-Apiose in 3, 4, 8–12, and 14–19, L-arabinose in 1–20, D-fucose in 4, 7, 10, 13, 15, and 16, D-galactose in 11–15, D-glucose in 5 and 13–15, D-quinovose in 2, L-rhamnose in 1–20, and D-xylose in 1–20 were identified by comparing their retention times (t_R) and optical rotations with those of authentic samples: D-apiose (6.66, positive optical rotation), L-rhamnose (7.64, negative optical rotation), D-fucose (8.42, positive optical rotation), D-quinovose (8.78, positive optical rotation), L-arabinose (9.57, positive optical rotation), D-xylose (9.92, positive optical rotation), D-galactose (15.63, positive optical rotation), and D-glucose (16.14, positive optical rotation).

Enzymatic Hydrolysis of 21

Compound 21 (15.0 mg) was treated with naringinase (45.0 mg, EC 232-962-4, Sigma) in AcOH/AcOK buffer (pH 4.3, 5.0 mL) at 28°C for 84 h. The reaction mixture was purified by silica gel CC using CHCl₃/MeOH/H₂O (90 : 10 : 1) to yield 21a (12.2 mg) and a sugar fraction (1.3 mg). HPLC analysis of the sugar fraction under the same conditions as those of 1 showed the presence of D-glucose (16.14, positive optical rotation).

Cytotoxic Activity Assay

HL-60 and A549 cells were kept in RPMI-1640 medium and MEM, respectively. These cell media contained 100 µg/mL streptomycin sulfate, penicillin G sodium salt, and heat-inactivated 10% FBS supplemented with L-glutamine. Cytotoxic activity of the isolated compounds against HL-60 and A549 cells was evaluated by a modified MTT assay method as previously reported.²⁴⁾

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) “The Grand Dictionary of Horticulture,” Vol. 4, ed. by Tsukamoto Y., Shogakukan, Tokyo, 1989, pp. 387–389.
• 2) Bassarello C., Muzashvili T., Skhirtladze A., Kemertelidze E., Pizza C., Piacente S., Phytochemistry, 69, 1227–1233 (2008).
• 3) Muzashvili T., Perrone A., Napolitano A., Kemertelidze E., Pizza C., Piacente S., Phytochemistry, 72, 2180–2188 (2011).
• 4) Kißmer B., Wichtl M., Planta Med., 52, 152–153 (1986).
• 5) Tsiftsoglou S. O., Stefanakis K. M., Lazari M. D., Biochem. Syst. Ecol., 79, 8–11 (2018).
• 6) Yang F. Y., Su Y. F., Wang Y., Chai X., Han X., Wu Z. H., Gao X. M., Biochem. Syst. Ecol., 38, 759–763 (2010).
• 7) Yang J., Zhang Y. H., Miao F., Zhou L., Sun W., Fitoterapia, 81, 636–639 (2010).
• 8) Zhang H., Su Y., Yang F., Zhao Z., Gao X., Phytochem. Lett., 10, 164–167 (2014).
• 9) Meng Y., Whiting P., Šik V., Rees H. H., Dinan L., Phytochemistry, 57, 401–407 (2001).
• 10) Watanabe K., Mimaki Y., Sakagami H., Sashida Y., J. Nat. Prod., 66, 236–241 (2003).
• 11) Yokosuka A., Iguchi T., Kawahata R., Mimaki Y., Phytochem. Lett., 23, 94–99 (2018).
• 12) Mimaki Y., Watanabe K., Sakuma S., Sakagami H., Sashida Y., Helv. Chim. Acta, 86, 398–407 (2003).
• 13) Watanabe K., Sakagami H., Mimaki Y., Heterocycles, 65, 775–785 (2005).
• 14) Mimaki Y., Matsuo Y., Watanabe K., Sakagami H., J. Nat. Med., 64, 452–459 (2010).
• 15) Zhang H., Su Y. F., Yang F. Y., Zhao Z. Q., Gao X. M., Helv. Chim. Acta, 97, 1652–1665 (2014).
• 16) Zhang H., Su Y. F., Yang F. Y., Nat. Prod. Res., 30, 1724–1730 (2016).
• 17) Zhang H., Su Y. F., Yang F. Y., Phytochem. Lett., 18, 213–219 (2016).
• 18) Zhang H., Su Y. F., Yang F. Y., Gao X. M., Nat. Prod. Res., 31, 925–931 (2017).
• 19) Asano T., Murayama T., Hirai Y., Shoji J., Chem. Pharm. Bull., 41, 566–570 (1993).
• 20) Mimaki Y., Inoue T., Kuroda M., Sashida Y., Phytochemistry, 43, 1325–1331 (1996).
• 21) Yang Q. X., Zhang Y. J., Li H. Z., Yang C. R., Steroids, 70, 732–737 (2005).
• 22) Watanabe Y., Sanada S., Ida Y., Shoji J., Chem. Pharm. Bull., 31, 3486–3495 (1983).
• 23) Iguchi T., Kuroda M., Naito R., Watanabe T., Matsuo Y., Yokosuka A., Mimaki Y., Molecules, 22, e1243 (2017).
• 24) Iguchi T., Kuroda M., Naito R., Watanabe T., Matsuo Y., Yokosuka A., Mimaki Y., J. Nat. Med., 73, 131–145 (2019).