Cycloartane and Oleanane Glycosides from the Tubers of Eranthis cilicica

Abstract

Phytochemical analysis of the tubers of Eranthis cilicica was performed as part of our continuous study on the plants of the family Ranunculaceae, which resulted in the isolation of eleven new cycloartane glycosides (1–11) and one new oleanane glycoside (13), together with one known oleanane glycoside (12). The structures of the new compounds were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis followed by either X-ray crystallographic or chromatographic analysis. The aglycone (1a) of 2 and its C-23 epimer (8a), and the oleanane glycosides (12 and 13) showed cytotoxic activity against HL-60 leukemia cells with IC₅₀ values ranging from 10.6 μM to 101.6 μM. HL-60 cells were much more sensitive to 8a (IC₅₀ 14.8 μM) than 1a (IC₅₀ 101.1 μM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. Compound 12 was revealed so as to partially induce apoptotic cell death in HL-60 cells, as was evident from morphology of HL-60 cells treated with 12.

1. Introduction

We carried out systematic phytochemical screenings of the plants belonging to the family Ranunculaceae, such as the Adonis [1,2,3,4], Anemone [5,6], Cimicifuga [7,8], Clematis [9], Helleborus [10,11,12,13,14], and Pulsatilla species [15,16], and isolated various triterpene and steroidal glycosides, including cardiac and pregnane glycosides. Among them, cycloartane glycosides had a unique structure and showed relationships between chemical structure and cytotoxic activity [8]. The genus Eranthis also belongs to the family Ranunculaceae and is taxonomically related to the genus Helleborus [17]. Previously, we have isolated two new oleanane bisdesmosides, eranthisaponins A and B [18], and eight new chromone derivatives from the tubers of Eranthis cilicica [19]. Further phytochemical examination of the E. cilicica tubers resulted in the isolation of eleven new cycloartane glycosides and one new oleanane glycoside, together with one known oleanane glycoside. We report herein the structural determination of the new compounds by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis, followed by either X-ray crystallographic or chromatographic analysis. As part of our ongoing phytochemical study of Ranunculaceae plants, the cytotoxic activity of the cycloartane-type glycosides 2 and 8, the aglycone 1a and its C-23 epimer 8a, and the oleanane-type triterpene glycosides 12 and 13 against HL-60 human promyelocytic leukemia cells is evaluated and briefly discussed.

2. Results and Discussion

2.1. Isolation and Structure Elucidation of 1–13

The MeOH extract of the E. cilicica tubers was allowed to pass through the porous-polymer polystyrene resin (Diaion HP-20) column, and a series of chromatographic separations of the glycoside-enriched fraction using column chromatography (CC) on silica gel and octadecylsilanized (ODS) silica gel were performed to obtain compounds 1–13 (Figure 1). The known compound 12 was identified as 3β-[(O-β-d-glucopyranosyl-(1→4)-O-[α-l-rhamnopyranosyl-(1→2)]-α-l-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid by direct comparison with an authentic sample isolated from Anemone coronaria [6].

Compound 1 was obtained as an amorphous solid, and its molecular formula was determined to be C₃₆H₅₆O₁₀, based on high-resolution (HR)-electrospray ionization (ESI)-time-of-flight (TOF)-MS (m/z 671.3794 [M + Na]⁺) data and ¹³C-NMR spectrum with 36 carbon signals (

Table 1. ¹³C-NMR spectral data for 1, 1a, 2–8, 8a, and 9–11 in C₅D₅N.

C	1	1a	2	3	4	5	6	7	8	8a	9	10	11
1	32.1	32.5	32.1	32.0	32.1	32.0	31.3	32.1	32.1	32.4	32.1	32.1	32.0
2	29.8	31.2	29.7	29.6	29.9	29.7	29.5	30.2	29.8	31.1	29.8	30.0	29.9
3	88.6	77.9	88.7	88.6	88.4	88.7	88.3	88.5	88.7	77.8	88.7	88.4	88.5
4	41.2	41.0	41.1	41.0	41.1	41.1	41.0	41.2	41.2	41.0	41.2	41.2	41.1
5	47.5	47.5	47.5	47.4	47.3	47.4	46.4	47.3	47.5	47.4	47.5	47.4	47.2
6	21.1	21.5	21.1	21.0	21.1	21.0	20.0	20.8	21.2	21.5	21.2	21.2	20.8
7	27.4	27.6	27.4	27.3	27.4	27.4	26.7	26.2	27.5	27.6	27.5	27.5	26.2
8	48.3	48.5	48.3	48.3	48.4	48.3	41.9	47.5	48.5	48.7	48.5	48.5	47.6
9	19.7	19.8	19.8	19.7	19.7	19.7	19.4	19.6	19.8	19.8	19.8	19.8	19.5
10	26.6	27.0	26.6	26.5	26.5	26.5	27.6	26.5	26.7	26.9	26.6	26.7	26.5
11	27.3	27.4	27.2	27.2	27.1	27.1	27.6	26.3	27.2	27.2	27.2	27.1	26.1
12	32.6	32.7	32.6	32.5	32.5	32.5	33.6	33.2	32.6	32.6	32.6	32.6	33.1
13	45.5	45.5	45.4	45.4	45.4	45.4	46.5	46.3	45.3	45.3	45.3	45.3	46.3
14	51.5	51.6	51.5	51.5	51.5	51.5	64.0	44.7	51.6	51.6	51.6	51.6	44.5
15	38.2	38.3	38.2	38.1	38.1	38.1	36.1	44.4	37.6	37.6	37.6	37.6	43.8
16	75.3	75.4	75.3	75.2	75.3	75.3	74.2	75.2	73.7	73.7	73.7	73.7	73.1
17	56.9	57.0	56.9	56.7	56.8	56.8	57.1	56.6	57.1	57.1	57.1	57.1	56.7
18	22.0	22.1	22.0	21.9	22.0	22.0	20.5	20.6	22.0	22.0	22.0	22.0	20.5
19	30.6	30.9	30.6	30.5	30.6	30.5	28.1	30.0	30.6	30.8	30.6	30.7	30.1
20	23.9	23.9	23.9	23.8	23.8	23.8	23.1	23.7	26.5	26.4	26.5	26.4	26.2
21	20.7	20.7	20.7	20.6	20.6	20.6	20.9	20.7	20.5	20.4	20.5	20.4	20.3
22	37.7	37.8	37.7	37.6	37.6	37.6	36.8	37.6	36.8	36.7	36.8	36.7	36.5
23	106.1	106.1	106.1	106.0	106.1	106.1	106.2	106.1	106.1	106.1	106.1	106.1	106.0
24	62.0	62.1	62.0	62.0	62.0	62.0	62.1	62.0	63.4	63.4	63.4	63.4	63.3
25	62.4	62.4	62.4	62.4	62.4	62.4	62.4	62.5	63.1	63.1	63.1	63.1	63.1
26	67.9	68.0	67.9	67.9	67.9	67.9	68.1	68.0	68.7	68.6	68.7	68.6	68.6
27	14.2	14.2	14.2	14.1	14.2	14.1	14.2	14.2	13.7	13.7	13.7	13.7	13.7
28	63.5	63.6	63.4	63.3	63.4	63.3	210.2	19.7	63.2	63.2	63.2	63.2	19.6
29	25.7	26.1	25.6	25.5	25.5	25.5	25.6	25.7	25.7	26.0	25.6	25.6	25.6
30	15.4	14.8	15.4	15.3	15.3	15.3	15.1	15.4	15.4	14.8	15.4	15.4	15.3
1′	106.8		106.4	106.1	106.6	106.3	106.4	106.8	106.4		106.2	106.7	106.6
2′	75.8		75.2	74.4	75.5	75.0	75.3	75.6	75.3		74.4	75.6	75.5
3′	78.7		76.9	88.7	78.2	76.5	77.0	78.5	76.9		88.9	78.5	78.5
4′	71.8		81.6	69.7	71.6	81.1	81.6	71.7	81.6		69.8	71.6	71.6
5′	78.3		76.2	77.7	77.0	74.9	76.3	77.1	76.2		77.9	77.1	77.0
6′	63.0		62.3	62.4	70.2	68.7	62.4	70.3	62.4		62.5	70.3	70.1
1′′			104.9	105.7	105.2	104.7	105.0	105.4	105.0		105.9	105.3	105.2
2′′			74.8	75.4	75.1	75.1	74.8	74.7	74.8		75.5	75.2	75.1
3′′			78.2	78.1	78.4	78.2	78.2	78.4	78.2		78.2	78.3	78.2
4′′			71.5	71.4	71.6	71.6	71.5	71.7	71.5		71.6	71.7	71.5
5′′			78.4	78.6	78.3	78.1	78.5	78.4	78.4		78.7	78.4	78.3
6′′			62.3	62.3	62.5	62.3	62.5	62.7	62.4		62.5	62.6	62.5
1′′′						105.0
2′′′						75.1
3′′′						78.2
4′′′						71.5
5′′′						78.1
6′′′						62.5

). The IR spectrum of 1 showed an absorption band attributed to hydroxy groups at 3388 cm⁻¹. The ¹H-NMR spectrum of 1 was typical of a triterpene glycoside based on a cycloartane derivative, showing signals for a cyclopropane methylene group at δ_H 0.48 and 0.20 (each d, J = 4.0 Hz); four tertiary methyl groups at δ_H 1.42, 1.36, 1.29, and 1.03 (each s); a secondary methyl group at δ_H 0.97 (d, J = 6.5 Hz); and an anomeric proton δ_H 4.94 (d, J = 7.8 Hz). Enzymatic hydrolysis of 1 using naringinase gave a new triterpene aglycone (1a: C₃₀H₄₆O₅) as colorless needles and d-glucose as a carbohydrate moiety. Based on X-ray crystallographic analysis, the structure of 1a was unambiguously established as (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-9,19-cycloartane-3β,28-diol (Figure 2). Linkage of a β-d-glucopyranosyl group to the C-3 hydroxy group of the aglycone in 1 was ascertained by long-range correlations between the anomeric proton (H-1′) at δ_H 4.94 and the C-3 carbon of the aglycone at δ_C 88.6 in the ¹H-detected heteronuclear multiple-bond connectivity (HMBC) spectrum of 1. Thus, 1 was established as (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl β-d-glucopyranoside.

Compound 2 had the molecular formula C₄₂H₆₆O₁₅, as determined by HR-ESI-TOF-MS (m/z 833.4297 [M + Na]⁺) and ¹³C-NMR (42 carbon signals) data. The molecular formula was larger than that of 1 by C₆H₁₀O₅, which corresponded to one hexosyl unit. The ¹H-NMR spectrum of 2 showed signals due to two anomeric protons at δ_H 5.20 (d, J = 7.9 Hz) and 4.87 (d, J = 7.8 Hz), as well as a cyclopropane methylene group at δ_H 0.47 and 0.20 (each d, J = 4.0 Hz), four tertiary methyl groups at δ_H 1.42, 1.35, 1.27, and 1.02 (each s), a secondary methyl group at δ_H 0.97 (d, J = 6.6 Hz). Enzymatic hydrolysis of 2 with naringinase gave 1a and d-glucose. The ¹H- and ¹³C-NMR signals of the diglycoside moiety, which were assigned based on ¹H-¹H correlation spectroscopy (COSY) and ¹H-detected heteronuclear multiple coherence (HMQC) spectra, indicated the presence of a terminal β-d-glucopyranosyl unit [δ_H 5.20 (d, J = 7.9 Hz, H-1′′ of Glc (II)); δ_C 104.9 (CH), 74.8 (CH), 78.2 (CH), 71.5 (CH), 78.4 (CH), 62.3 (CH₂)] and a C-4 glycosylated β-d-glucopyranosyl unit [δ_H 4.87 (d, J = 7.8 Hz, H-1′ of Glc (I)); δ_C 106.4 (CH), 75.2 (CH), 76.9 (CH), 81.6 (CH), 76.2 (CH), 62.3 (CH₂)] in the molecule of 2 [20]. In the HMBC spectrum of 2, long-range correlations were observed between H-1′′ of Glc (II) and C-3′ of Glc (I), and between H-1′ of Glc (I) and C-3 of the aglycone moiety at δ_C 88.7. The molecular formula of 2 was assigned as (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cylcoartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside.

Compounds 3 and 4, which had the same molecular formula as 2 (C₄₂H₆₆O₁₅), gave 1a and d-glucose upon enzymatic hydrolysis. The ¹H- and ¹³C-NMR spectra of 3 and 4 suggested that they were constitutional isomers of 2 based on the linkage position of the terminal d-glucosyl unit (Glc (II)) at the inner d-glucosyl unit (Glc (I)) in the diglucosyl residue. In the HMBC spectrum of 3, H-1′′ of Glc (II) at δ_H 5.28 (d, J = 7.9 Hz) showed a long-range correlation with C-3′ of Glc (I) at δ_C 88.7, and H-1′ of Glc (I) at δ_H 4.86 (d, J = 7.8 Hz), in turn, showed a long-range correlation with C-3 of the aglycone at δ_C 88.6. On the other hand, HMBC correlations were observed between H-1′′ of Glc (II) at δ_H 5.28 (d, J = 7.8 Hz) and C-6′ of Glc (I) at δ_C 70.2, and between H-1′ of Glc (I) at δ_H 4.86 (d, J = 7.7 Hz) and C-3 of the aglycone at δ_C 88.4 in 4. Compounds 3 and 4 were established as O-β-d-glucopyranosyl-(1→3)-β-d-glucopyranoside and O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside of (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl, respectively.

Compound 5 (C₄₈H₇₆O₂₀) also yielded 1a and d-glucose upon enzymatic hydrolysis. The ¹H- and ¹³C-NMR spectra of 5 showed signals for two terminal β-d-glucopyranosyl units [δ_H 5.45 (d, J = 7.9 Hz, H-1′′ of Glc (II)); δ_C 104.7 (CH), 75.1 (CH), 78.2 (CH), 71.6 (CH), 78.1 (CH), 62.3 (CH₂); and δ_H 5.34 (d, J = 7.8 Hz, H-1′′′ of Glc (III)); δ_C 105.0 (CH), 75.1 (CH), 78.2 (CH), 71.5 (CH), 78.1 (CH), 62.5 (CH₂)] and a C-4 and C-6 diglycosylated β-d-glucopyranosyl unit [δ_H 4.80 (d, J = 7.5 Hz, H-1′ of Glc (I)); δ_C 106.3 (CH), 75.0 (CH), 76.5 (CH), 81.1 (CH), 74.9 (CH), 68.7 (CH₂)] [21]. In the HMBC spectrum of 5, long-range correlations were observed between H-1′′ of Glc (II) and C-4′ of Glc (I), H-1′′′ of Glc (III) and C-6′ of Glc (I), and between H-1′ of Glc (II) and C-3 of the aglycone at δ_C 88.7. Thus, 5 was deduced to be (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cylcoartan-3β-yl O-β-d-glucopyranosyl-(1→4)-O-[β-d-glucopyranosyl(1→6)]-β-d-glucopyranoside.

Compounds 6 and 7 had the molecular formula C₄₂H₆₄O₁₅ and C₄₂H₆₆O₁₄, respectively, based on HR-ESI-TOF-MS and ¹³C-NMR data. The ¹H- and ¹³C-NMR spectral properties of 6 were essentially analogous to those of 2; however, the hydroxymethyl signals at δ_H 4.02 and 3.94 (each d, J = 11.5 Hz) and δ_C 63.4 assignable to H₂-28 and C-28 in 2 were replaced by those due to an aldehyde group at δ_H 10.14 (s) and δ_C 210.2 in 6. Treatment of 6 with NaBH₄ in EtOH afforded 2. On the other hand, comparison of the ¹H- and ¹³C-NMR spectra of 7 with those 4 revealed that the H₂-28/C-28 hydroxymethyl proton and carbon signals observed for 4 disappeared in the case of 7. Instead, the signals from a tertiary methyl group were detected at δ_H 0.80 (s) and δ_C 19.7 in the spectrum of 7. The methyl proton signal showed HMBC correlations with the C-8 (δ_C 47.5), C-13 (δ_C 46.3), C-14 (δ_C 44.7) and C-15 (δ_C 44.4) carbon signals. Compounds 6 and 7 were determined to be (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-28-oxo-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside and (23R,24R,25R)-16β,23:23,26:24,25-triepoxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside.

Compound 8 had the same molecular formula as 2 (C₄₂H₆₆O₁₅), and its ¹H- and ¹³C-NMR spectral features were closely related to those of 2, except for the signals from the ring E and F parts. Enzymatic hydrolysis of 8 gave an aglycone (8a; C₃₀H₄₆O₅) and d-glucose. The phase-sensitive NOE correlation spectroscopy (PHNOESY) spectrum of 8a showed NOE correlations between H-16 (δ_H 4.80) and H-22 α (δ_H 1.56)/H-26a (δ_H 3.82), H-20 (δ_H 1.85) and H-24 (δ_H 3.69), and between H-22β(δ_H 2.16) and H-24, indicating that 8a was a new aglycone, the C-23 epimer of 1a. Compound 8 was assigned as (23S,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside.

Compounds 9 and 10 had the same molecular formula C₄₂H₆₆O₁₅, and their ¹H- and ¹³C-NMR spectra were suggestive of cycloartane diglucosides closely related to 8. Indeed, enzymatic hydrolysis of 9 and 10 gave 8a and d-glucose. Assignments of the ¹H- and ¹³C-NMR signals from the sugar moieties of 9 and 10, which were established by ¹H-¹H COSY and HMQC spectral analysis, implied that the diglucoside sequences of 9 and 10 corresponded to those of 3 and 4, respectively. Furthermore, HMBC correlations were observed from H-1′′ of Glc (II) at δ_H 5.31 (d, J = 7.9 Hz) to C-3′ of Glc (I) at δ_C 88.9, and from H-1′ at δ_H 4.89 (d, J = 7.8 Hz) to C-3 of the aglycone at δ_C 88.7 in 9, and from H-1′′ of Glc (II) at δ_H 5.16 (d, J = 7.8 Hz) to C-6′ of Glc (I) at δ_C 70.3, and from H-1′ at δ_H 4.89 (d, J = 7.7 Hz) to C-3 of the aglycone at δ_C 88.4 in 10. Compounds 9 and 10 were formulated as O-β-d-glucopyranosyl-(1→3)-β-d-glucopyranoside and O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside of (23S,24R,25R)-16β,23:23,26:24,25-triepoxy-28-hydroxy-9,19-cylcoartan-3β-yl, respectively.

Compound 11 (C₄₂H₆₆O₁₄) bore close similarity to 10 in terms of the ¹H- and ¹³C-NMR spectral features. However, the hydroxymethyl signals at δ_H 3.98 and 3.89 (each d, J = 11.5 Hz) and δ_C 63.2 assignable to H₂-28 and C-28 in 10 were replaced by the signals due to a methyl group at δ_H 0.79 (s) and δ_C 19.6 in 11. In addition, the methyl proton signal showed HMBC correlations with the C-8 (δ_C 47.6), C-13 (δ_C 46.3), C-14 (δ_C 44.5), and C-15 (δ_C 43.8) carbon signals. Compound 11 was established to be (23S,24R,25R)-16β,23:23,26:24,25-triepoxy-9,19-cycloartan-3β-yl O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranoside.

Compound 13 had the molecular formula C₅₃H₈₆O₂₂, as revealed by HR-ESI-TOF-MS (m/z 1075.5710 [M + H]⁺) and ¹³C-NMR (53 carbon signals) data. The molecular formula was larger than that of 12 by C₆H₁₀O₅, which corresponded to one hexosyl unit. The ¹H-NMR spectrum of 13 displayed signals for four anomeric protons at δ_H 6.02 (br s), 5.29 (d, J = 7.8 Hz), 5.06 (d, J = 7.9 Hz) and 4.92 (d, J = 6.9 Hz), as well as the signals for six tertiary methyl groups at δ_H 1.25, 1.05, 1.02, 1.00, 0.94 and 0.92 (each s), and an olefinic proton at δ_H 5.46 (t-like, J = 3.0 Hz), suggesting that this compound was a tetraglycoside of an oleanoic acid derivative. Acid hydrolysis of 13 with 1M HCl in dioxane-H₂O (1:1) gave 23-hydroxyolean-12-en-28-oic acid (hederagenin) as the aglycone [22], and l-arabinose, d-galactose, d-glucose and l-rhamnose as the carbohydrate moieties. When the ¹³C-NMR spectrum of 13 was compared with that of 12 [6], six signals assignable to a terminal β-d-galactopyraosyl group (Gal) were observed in addition to signals for a 2,4-disubstituted α-l-arabinopyranosyl group (Ara), C-3 substituted α-l-rhamnopyranosyl group (Rha), and terminal β-d-glucopyranosyl group (Glc). The anomeric configurations of Ara, Gal and Glc were confirmed to be α, β, and β, respectively, based on the relatively large ³J_H-1,H-2 values (6.9–7.9 Hz). In the case of the Rha moiety, the large ¹J_C-1,H-1 (172 Hz) was indicative of the α-anomeric configuration [6]. In the HMBC spectrum of 13, long-range couplings were observed between H-1′′′ of Glc (δ_H 5.29) and C-4′ of Ara at δ_C 80.0, H-1′′′′ of Gal (δ_H 5.06) and C-3′′ of Rha at δ_C 83.2, H-1′′ of Rha (δ_H 6.02) and C-2′ of Ara at δ_C 76.5, and between H-1′ of Ara (δ_H 4.92) and C-3 of the aglycone at δ_C 81.3. Compound 13 was determined to be 3 β-[(O-β-d-galactopyranosyl-(1→3)-O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-glucopyranosyl-(1→4)]-α-l-arabinopyranosyl)oxy]-23-hydroxyolean-12-en-28-oic acid.

2.2. Cytotoxic Activity of 1a, 2, 8, 8a, 12, and 13

We previously reported that the slight differences in the structure of cycloartane glycosides effected on cytotoxic activity [8]. In this study, some selected compounds, the new cycloartane glycosides (2 and 8), and the aglycone (1a) of 2 and its C-23 epimer (8a), as well as oleanane glycosides (12 and 13) were evaluated for their cytotoxic activity against HL-60 cells using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (

Table 2. Cytotoxic activity of 1a, 2, 8, 8a, 12, 13, and etoposide against HL-60 cells ^a.

Compound	IC50 (µM)
1a	101.1 ± 0.44
2	>200
8	>200
8a	14.8 ± 1.00
12	10.6 ± 0.40
13	10.8 ± 0.53
etoposide	0.32 ± 0.01

^a Data represent the mean value ± standard error of the mean (SEM) of three experiments performed in triplicate.

). Etoposide was used as a positive control, and it gave an IC₅₀ value of 0.32 ± 0.01 μM. The cytotoxic activity of 8a against HL-60 cells (IC₅₀ 14.8 ± 1.00 μM) was much more potent than that of 1a (IC₅₀ 101.1 ± 0.44 μM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. On the other hand, 2 and 8 were not cytotoxic to HL-60 cells at sample concentrations up to 200 μM. Compounds 12 and 13 were cytotoxic to HL-60 cells, with IC₅₀ values of 10.6 ± 0.40 μM and 10.8 ± 0.53 μM, respectively. After HL-60 cells were exposed to 12 at a sample concentration of 20 μM for 72 h, they were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and observed under a fluorescence microscope. The cells showed nuclear chromatin condensation and nuclear disassembly, illustrated in Figure 3. Therefore, 12 partially induced apoptotic cell death in HL-60 cells.

3. Materials and Methods

3.1. General Experimental Procedures and Plant Material

The instruments, experimental conditions, and plant material used (except for those mentioned below) were the same as those described in previous papers [18,19]. Melting point was determined on an MP-3 melting point apparatus (Yanaco, Kyoto, Japan). X-ray diffraction experiments were carried out on a DIP image plate diffractometer (Bruker AXS, Karlsruhe, Germany).

3.2. Extraction and Isolation

The MeOH extract (135 g) of Eranthis cilicica tubers (1.3 kg) was subjected to Diaion HP-20 CC [19]. The 80% MeOH eluted portion (30 g) was chromatographed on silica gel eluted with gradient mixtures of CHCl₃-MeOH (20:1; 9:1, 4:1, 2:1) and finally with MeOH to give 9 subfractions (Frs. A–I). Fr. E was subjected silica gel CC eluted with CHCl₃-MeOH (9:1) and ODS silica gel CC eluted with MeCN-H₂O (1:2) to yield 1 (17.0 mg) and 7 (13.4 mg). Fr. G was repeatedly subjected to silica gel CC eluted with CHCl₃-MeOH-H₂O (60:10:1, 50:10:1, 40:10:1) and ODS silica gel CC eluted with MeCN-H₂O (5:8, 5:9, 1:2) to yield 2 (121 mg), 3 (7.8 mg), 8 (100 mg), 9 (4.1 mg), 10 (35.0 mg), and 11 (5.1 mg). Fr. H was subjected to CC on silica gel eluted with CHCl₃-MeOH-H₂O (30:10:1) and ODS silica gel eluted with MeCN-H₂O (1:2) and MeOH-H₂O (7:5) to yield 13 (15.2 mg). Fr. I was subjected to CC on silica gel eluted with CHCl₃-MeOH-H₂O (14:8:1; 10:10:1) and ODS silica gel eluted with MeCN-H₂O (5:8, 2:5, 4:11, 1:3) to yield 12 (13.7 mg). The 50% MeOH eluted portion (35 g) was chromatographed on silica gel eluted with CHCl₃-MeOH-H₂O (40:10:1) to give 6 subfractions (Frs. a–f). Fr. b was subjected to silica gel column eluted with CHCl₃-MeOH (20:1) and CHCl₃-MeOH-H₂O (40:10:1) and ODS silica gel column eluted with MeCN-H₂O (1:2, 2:5, 1:3) to yield 4 (15.2 mg) and 6 (10.0 mg). Fr. c was subjected to silica gel CC eluted with CHCl₃-MeOH (10:1) and CHCl₃-MeOH-H₂O (40:10:1) and ODS silica gel CC eluted with MeCN-H₂O (1:2) to yield 5 (33.8 mg).

3.3. Structural Characterization

Compound 1: Amorphous solid. [α]_D²⁵ −46.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 671.3794 [M + Na]⁺ (Calcd for C₃₆H₅₆NaO₁₀, 671.3771). IR ν_max (film) cm⁻¹: 3388 (OH), 2928 and 2870 (CH). ¹H-NMR spectral data for 1 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Enzymatic Hydrolysis of 1 Compound 1 (4.5 mg) was treated with naringinase (EC 232-96-4, Sigma; 22.5 mg) in a mixture of HOAc/KOAc buffer (pH 4.3, 5 mL) and EtOH (2 mL) at room temperature for 72 h. The reaction mixture was purified by silica gel CC eluted with CHCl₃-MeOH (22:1) followed by MeOH to give 1a (2.4 mg) and a sugar fraction (0.6 mg). The sugar fraction was analyzed by HPLC under the following conditions: Capcell Pak NH₂ UG80 column (4.6 mm i.d. × 250 mm, 5 μm, Shiseido, Tokyo, Japan); mobile phase of MeCN-H₂O (7:3); detection by refractive index and optical rotation, and at a flow rate of 1.0 mL/min. d-glucose was identified by comparing its retention time and optical rotation with those of an authentic sample; t_R 11.62 min (d-glucose, positive optical rotation).

Compound 1a: Colorless needles from MeOH-MeCN (1:1). mp 281–285 °C. [α]_D²⁴ −48.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 509.3255 [M + Na]⁺ (Calcd for C₃₀H₄₆NaO₅, 509.3243). IR ν_max (film) cm⁻¹: 3476 (OH), 2953, 2925 and 2868 (CH). ¹H-NMR spectral data for 1a are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

X-Ray Crystallography of 1a Monoclinic, space group C2, unit cell dimension a = 32.945(2) Å, b = 6.9250(2) Å, c = 13.5690(6), β = 111.270(2)°, V = 2884.8(2) ų, Z = 4; T = 296 K, d_calc = 1.204 Mg/m³; μ (Mo Kα, λ = 0.71073 Å) = 0.084 mm⁻¹; R [I > 2σ(I)] = 0.0370, wR [I > 2σ(I)] = 0.1054, R [for all data] = 0.0409, wR [for all data] = 0.1081. Crystallographic data of 1a have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as supplementary publication no. CCDC 1877743. Copies of the data can be obtained free of charge from the CCDC via http://beta-www.ccdc.cam.ac.uk.

Compound 2: Amorphous solid. [α]_D²⁶ −34.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 833.4297 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3397 (OH), 2930 and 2870 (CH). ¹H-NMR spectral data for 2 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 3: Amorphous solid. [α]_D²⁴ −48.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 833.4359 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3450 (OH), 2932 and 2872 (CH). ¹H-NMR spectral data for 3 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 4: Amorphous solid. [α]_D²⁶ −44.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 833.4340 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3408 (OH), 2933 and 2871 (CH). ¹H-NMR spectral data for 4 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 5: Amorphous solid. [α]_D²⁸ −16.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 973.5001 [M + H]⁺ (Calcd for C₄₈H₇₇O₂₀, 973.5008). IR ν_max (film) cm⁻¹: 3363 (OH), 2932 and 2872 (CH). ¹H-NMR spectral data for 5 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Enzymatic Hydrolysis of 2–5 Compounds 2 (54.1 mg), 3 (2.5 mg), 4 (5.1 mg), and 5 (4.8 mg) were independently subjected to enzymatic hydrolysis with naringinase as described for 1 to give 1a (25.2 mg from 2; 1.3 mg from 3; 2.5 mg from 4; 2.6 mg from 5) and a sugar fraction (5.9 mg from 2; 0.4 mg from 3; 0.8 mg from 4; 1.2 mg from 5). HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1 showed the presence of d-glucose.

Compound 6: Amorphous solid. [α]_D²⁴ −84.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 831.4100 [M + Na]⁺ (Calcd for C₄₂H₆₄NaO₁₅, 831.4143). IR ν_max (film) cm⁻¹: 3387 (OH), 2925 and 2870 (CH), 1712 (C=O). ¹H-NMR spectral data for 6 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Preparation of 2 from 6 Compound 6 (1.7 mg) was dissolved in NaBH₄ (2.2 mg) ethanolic solution (1 mL) and it was stirred at room temperature for 5 h. After Me₂CO was added to the reaction mixture, the solvent was removed under reduced pressure. The residue was suspended with H₂O (3 mL) and extracted with EtOAc (3 mL × 3). The EtOAc extract was chromatographed on silica gel eluted with CHCl₃-MeOH-H₂O (30:10:1) to yield 2 (1.2 mg).

Compound 7: Amorphous solid. [α]_D²⁵ −70.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 817.4393 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₄, 817.4350). IR ν_max (film) cm⁻¹: 3418 (OH), 2931 and 2868 (CH). ¹H-NMR spectral data for 7 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 8: Amorphous solid. [α]_D²⁶ −50.0 (c 0.10, C₅H₅N). HR-ESI-TOF-MS m/z: 833.4359 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3348 (OH), 2926 and 2867 (CH). ¹H-NMR spectral data for 8 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 8a: Amorphous solid. [α]_D²⁴ −58.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 487.3423 [M + H]⁺ (Calcd for C₃₀H₄₇O₅, 487.3424). IR ν_max (film) cm⁻¹: 3442 (OH), 2955, 2927 and 2870 (CH). ¹H-NMR spectral data for 8a are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 9: Amorphous solid. [α]_D²⁶ −36.0 (c 0.10, C₅H₅N). HR-ESI-TOF-MS m/z: 833.4276 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3347 (OH), 2919 and 2871 (CH). ¹H-NMR spectral data for 9 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 10: Amorphous solid. [α]_D²⁶ −72.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 833.4313 [M + Na]⁺ (Calcd for C₄₂H₆₆NaO₁₅, 833.4299). IR ν_max (film) cm⁻¹: 3388 (OH), 2933 and 2872 (CH). ¹H-NMR spectral data for 10 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Enzymatic Hydrolysis of 8–10 Compounds 8 (31.3 mg), 9 (1.9 mg), and 10 (5.3 mg) were independently subjected to enzymatic hydrolysis with naringinase as described for 1 to give 8a (19.4 mg from 8; 0.9 mg from 9; 2.8 mg from 10) and a sugar fraction (3.2 mg from 8; 0.3 mg from 9; 1.1 mg from 10). HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1 showed the presence of d-glucose.

Compound 11: Amorphous solid. [α]_D²⁶ −62.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 795.4583 [M + H]⁺ (Calcd for C₄₂H₆₇O₁₄, 795.4531). IR ν_max (film) cm⁻¹: 3387 (OH), 2927 and 2871 (CH). ¹H-NMR spectral data for 11 are provided in the Supplementary Materials. ¹³C-NMR, see Table 1.

Compound 13: Amorphous solid. [α]_D²⁷ +6.0 (c 0.10, MeOH). HR-ESI-TOF-MS m/z: 1075.5710 [M + H]⁺ (Calcd for C₅₃H₈₇O₂₂, 1075.5689). IR ν_max (film) cm⁻¹: 3376 (OH), 2926 and 2858 (CH), 1696 (C=O). ¹H-NMR (C₅D₅N) δ_H 6.02 (1H, br s, H-1′′), 5.46 (1H, t-like, J = 3.0 Hz, H-12), 5.29 (1H, d, J = 7.8 Hz, H-1′′′), 5.06 (1H, d, J = 7.9 Hz, H-1′′′′), 4.92 (1H, d, J = 6.9 Hz, H-1′), 4.48 (1H, dd, J = 9.3, 7.8 Hz, H-2′′′), 4.46 (1H, br s, H-4′′′), 4.36 (1H, m, H-6a′′′), 4.34 (1H, m, H-6b′′′), 4.24 (1H, d, J = 11.0 Hz, H-23a), 4.18 (1H, dd, J = 10.5, 4.6 Hz, H-3), 4.10 (1H, dd, J = 9.3, 3.3 Hz, H-3′′′), 4.07 (1H, m, H-5′′′), 3.86 (1H, d, J = 11.0 Hz, H-23b), 3.26 (1H, dd, J = 13.8, 3.9 Hz, H-18), 1.54 (3H, d, J = 6.1 Hz, Me-6′′), 1.25 (3H, s, Me-27), 1.05 (3H, s, Me-24), 1.02 (3H, s, Me-26), 1.00 (3H, s, Me-30), 0.94 (3H, s, Me-25), 0.92 (3H, s, Me-29). ¹³C-NMR (C₅D₅N) δ_C: 39.0, 26.3, 81.3, 43.5, 47.6, 18.2, 32.9, 39.8, 48.2, 36.9, 23.8, 122.6, 144.8, 42.2, 28.3, 23.7, 46.7, 42.0, 46.5, 30.9, 34.2, 32.0, 64.0, 13.9, 16.0, 17.5, 26.1, 180.1, 33.2, 23.8 (aglycone C-1–C-30), 104.7, 76.5, 74.2, 80.0, 65.5 (Ara C-1–C-5), 101.6, 71.2, 83.2, 72.6, 69.9, 18.5 (Rha C-1–C-6), 107.1, 73.3, 75.2, 70.0, 76.8, 62.1 (Gal C-1–C-6), 106.5, 75.5, 78.3, 71.4, 78.5, 62.5 (Glc C-1–C-6).

Acid Hydrolysis of 13 A solution of 13 (5.0 mg) in 1 M HCl (dioxane-H₂O, 1:1, 3 mL) was heated at 95 °C for 1 h under an Ar atmosphere. After the reaction mixture was diluted with H₂O (2 mL), it was extracted with Et₂O (5 mL × 2). The Et₂O extract was chromatographed on silica gel eluted with CHCl₃-MeOH (19:1) to give hederagenin (2.6 mg). The H₂O residue was neutralized using an Amberlite IRA-93ZU (Organo, Tokyo, Japan) column and passed through a Sep-Pak C₁₈ cartridge (Waters, Milford, MA) eluted with H₂O-MeOH (3:2) to give a sugar fraction (1.8 mg). HPLC analysis of the sugar fraction under the same conditions as in the case of that of 1 (flow rate, 0.9 mL/min) showed the presence of l-arabinose, d-galactose, d-glucose, and l-rhamnose. t_R (min): 7.08 (negative optical rotation, l-rhamnose), 8.14 (positive optical rotation, l-arabinose), 12.86 (positive optical rotation, d-galactose), and 13.17 (positive optical rotation, d-glucose).

3.4. Cytotoxic Activity

HL-60 cells were maintained in an RPMI-1640 medium. The cell media contained heat-inactivated 10% (v/v) FBS supplemented with l-glutamine, penicillin G sodium salt (100 units/mL), and streptomycin sulfate (100 μg/mL). HL-60 (4 × 10⁴ cells/mL) cells were continuously treated with each compound for 72 h, and cell growth was measured using an MTT reduction assay as previously described [23]. Data represented as mean ± S.E.M. of three experiments performed in triplicate. The concentration, resulting in a 50% inhibition value (IC₅₀), was calculated from the dose response curve.

3.5. DAPI Staininig

The cells (1 × 10⁵ cells/mL) were plated on coverslips in 96-well plates. After 24 h, HL-60 cells were treated with either 20 μM of 12 or 17 μM of etoposide for 72 h. The cells were fixed with 1% glutaraldehyde for 30 min at room temperature before staining with DAPI (0.5 μg/mL in H₂O). They were observed under a CKX41 fluoroscence microscope (Olympus, Tokyo, Japan).

4. Conclusions

Further phytochemical examination of E. cilicica tubers gave eleven new cycloartane glycosides (1–11) and one new oleanane glycoside (13), together with one known oleanane glycosides (12). The structures of the new compounds were established by extensive spectroscopic analysis, including 2D NMR, and enzymatic hydrolysis, followed by either X-ray crystallographic or chromatographic analysis. The new cycloartane glycosides 2 and 8, the aglycone 1a and its C-23 epimer 8a, and oleanane-type triterpene glycosides 12 and 13 were evaluated for their cytotoxic activity against HL-60 leukemia cells. The cytotoxic activity of 8a was much more potent than that of 1a, indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. This is the first report of structure–activity relationships of cycloartane-type triterpenoids on C-23 epimer. Compounds 12 and 13 were moderately cytotoxic to HL-60 cells, and 12 partially induced apoptotic cell death in HL-60 cells.