Cycloartane and Oleanane Glycosides from the Tubers of Eranthis cilicica

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.


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.

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).

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 × 10 4 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 (IC 50 ), was calculated from the dose response curve.

DAPI Staininig
The cells (1 × 10 5 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 H 2 O). They were observed under a CKX41 fluoroscence microscope (Olympus, Tokyo, Japan).

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.