Compound 1 was obtained as a white amorphous solid. The molecular formula was determined to be C₄₈H₇₄O₂₀ from the molecular ion peak [M+Na]⁺ at m/z 993.4698 (calcd. for C₄₈H₇₄O₂₀Na, 993.4671) in the positive HRESI-MS. The analysis of the ¹H-NMR spectrum of 1, in combination with HSQC data, showed signals for three anomeric protons δ_H 4.95/δ_C 101.7, δ_H 4.50/δ_C 100.1 and δ_H 4.28/δ_C 103.1, suggesting the presence of three sugar moieties.

In addition, the ¹H-NMR spectrum (Table 1) showed signals for two tertiary methyl groups at δ_H 1.09 and 0.86 (each 3H, s) and one secondary methyl group at δ_H 1.02 (3H, d, 6.8 Hz), as well as one signal for a trisubstituted double bond (δ_H 5.55, 1H, br d, 5.6 Hz) and for an exometylene group (δ_H 5.09 and 4.92 each 1H, br s), three methine proton signals at δ_H 4.56 (1H, m, H-16), 3.38 (1H, m, H-1) and 3.33 (1H, m, H-3) indicative of secondary alcoholic functions and two methylene proton signals at δ_H 4.33 and 4.13 (each 1H, d, 12.5 Hz) ascribable to a primary alcoholic function.

The ¹³C-NMR spectrum showed three secondary alcoholic functions at δ_C 83.9, 81.8 and 68.6, one primary alcoholic function at δ_C 72.4 and a hemiacetalic carbon signal at δ_C 111.7, suggesting the presence of a furostanol skeleton (Table 1). Comparison with literature data and analysis of HSQC and HMBC data revealed a furosta-5,25(27)-diene-1β,3β,22α,26-tetrol moiety. Glycosylation shifts on the aglycone were observed for C-1 (δ_C 83.9) and C-26 (δ_C 72.4). The C-22 α-configuration of 1 was assigned on the basis of key ROESY correlations between H-20 (δ_H 2.14) and the protons H₂-23 (δ_H 1.84) and of the downfield shift of H-16 at δ_H 4.56 [9].

As concerning the sugar portion, in addition to the carbinol protons, the ¹H-NMR spectrum (Table 2) showed signals at δ_H 2.03 and 2.04 (each 3H, s), ascribable to the methyl groups of two acetyl groups [δ_C 170.8 and 170.9 (C=O); δ_C 20.7 and 20.8 (CH₃)], and one signal at δ_H 1.26 (3H, d, 6.3 Hz) indicative of a 6-deoxyhexopyranose unit (Table 2).

The assignment of all protons and carbon chemical shifts of the three sugar units was performed by careful analysis of 2D NMR spectra, including COSY, TOCSY, HSQC and HMBC experiments, allowing the identification of one β-glucopyranosyl (Glc), one α-arabinopyranosyl (Ara) and one α-rhamnopyranosyl (Rha) units. The relatively large J_H1-H2 values (7.4–8.0 Hz) indicated a β-orientation for the anomeric center of glucose and an α-orientation for that of arabinose in their pyranose form, whereas a small J_H1-H2 coupling (1.2 Hz) indicated the α-configuration of the rhamnopyranosyl unit. The monosaccharides obtained from the acidic hydrolysis of 1 were identified as D-glucose, L-arabinose and L-rhamnose by GC analysis of their chiral derivatives [10].

The position of the acetyl groups at C-3′ and C-4′ of the arabinose unit was suggested by the downfield shift observed for the H-3′ (δ_H 5.05) and H-4′ (δ_H 5.30) and for the upfield shift of C-2′ (δ_C 73.5) and C-5′ (δ_C 64.5) in comparison with the data reported for the authentic sample, ruscoponticoside E (6), which is known compound also isolated in the present study (Table 2). These evidences were confirmed by the HMBC correlations between the proton signals at δ_H 5.05 (H-3′) and δ_H 5.30 (H-4′) with the carbonyl resonances at 170.9 ppm and 170.8 ppm (Figure 3), respectively.

The sequence and interglycosidic linkages among the three sugar units and the aglycone were revealed by HMBC experiment (Figure 3). In the HMBC spectrum, a correlation peak between H-1′′′ of Glc at δ_H 4.28 and C-26 at δ_C 72.4, implied that the glucose unit is attached to C-26 of the aglycone, which is a structural feature in plant furostanol saponins. The linkage of the arabinose unit to C-1 of the aglycone was ascertained by the HMBC correlation between H-1′ of arabinose (δ_H 4.50) and C-1 at δ_C 83.9. Furthermore the anomeric proton of rhamnose at δ_H 4.95 was correlated with C-2′ of arabinose at δ_C 73.5 which supported the proposed sequence of the disaccharidic chain linked at C-1 of the aglycone.

Thus the structure of compound 1 was established as 26-O-β-D-glucopyranosylfurosta-5,25(27)-diene-1β,3β,22α,26-tetrol 1-O-[α-L-rhamnopyranosyl-(1′′→2′)-O-(3′,4′-di-O-acetyl)-α-L-arabino- pyranoside].

HRESI-MS of compound 2 showed a pseudomolecular ion peak at m/z 1007.4857 [M+Na]⁺ corresponding to the molecular formula C₄₉H₇₆O₂₀, which differs from that of compound 1 only in the gain of 14 u.m.a. ¹H-NMR [δ_H 3.16 (3H, s)] and ¹³C-NMR [δ_C 113.3 (C-22) and 47.5 (-OCH₃)] data suggested compound 2 to be a 22-methoxyfurostanol saponin (Table 1).

A HMBC experiment confirmed this hypothesis and showed a correlation between the methoxy group at δ_H 3.16 and C-22 (δ_C 113.3) of the aglycone. The ROESY experiment allowed us to assign the stereochemistry of the ketal carbon C-22. Clear correlations were observed between the methoxy group at δ_H 3.16 and the H-16 at δ_H 4.38 and between H-20 (δ_H 2.22) and H-23a (δ_H 1.90)/H-23b (δ_H 1.84) indicating an α-orientation of the methoxy group [3].

Analysis of COSY, HSQC and HMBC experiments revealed that 2 possessed sugar moieties identical to those of 1 (Table 2). Acidic hydrolysis of 2 afforded D-glucose, L-arabinose and L-rhamnose which were confirmed by GC analysis. Thus saponin 2 was elucidated as 26-O-β-D-glucopyranosyl-22α-methoxy-furosta-5,25(27)diene-1β,3β,26-triol 1-O-[α-L-rhamnopyranosyl-(1′′→2′)-O-(3′,4′-di-O-acetyl)-α-L-arabinopyranoside].

Although we have used mild extraction conditions (room temperature) we cannot exclude the possibility that compound 2 is an artifact due to reaction of compound 1 with the extraction solvent (MeOH).

Compound 3a showed the molecular formula of C₃₃H₅₁O₁₃S, deduced by a HRESI-MS measurement (m/z 687.3043, [M−H]⁻). The presence of a sulphate group was indicated by a fragment ion peak at m/z 631 [M−NaSO₃+H+Na]⁺ in the ESI-MS/MS spectrum recorded in a positive ion mode, corresponding to the loss of a SO₃Na from the parent ion, and by IR bands at 1245 and 1086 cm⁻¹.

Preliminary ¹H-NMR analysis of 3a (Table 3) indicated the steroid glycoside nature of the compound. The ¹H-NMR spectrum showed three methyl signals: two tertiary (δ_H 0.86 and 1.10) and one secondary (δ_H 1.04), and one anomeric proton signal at δ_H 4.28. Its ¹³C-NMR spectrum exhibited 33 carbon signals, with 27 being attributable to the aglycone and six attributable to the monosaccharide unit. The ¹³C-NMR spectrum further showed three secondary alcoholic functions at δ_C 85.5, 81.8 and 68.6, one primary alcoholic function at δ_C 72.6 and a hemiacetalic carbon signal at δ_C 111.1 indicating a furostane nature for the steroidal aglycone of 3a.

Combined analysis of COSY and TOCSY experiments allowed the detection of five spin systems, four belonging to the aglycone moiety and one attributable to the monosaccharide.

The location of the sulphate group at position-1 was inferred by the downfield shift of the corresponding nuclei (H-1, δ_H 4.03 and C-1, δ_C 85.5) [7]. The relative stereochemistry at C-1 and C-3 was evaluated by an accurate coupling constants analysis and by ROESY experiments. In particular, H-1 appeared as a double doublet (11.9 and 3.8 Hz), whereas H-3 appeared as a dddd with two large (ax-ax) and two small (ax-eq) coupling constants. These data pointed to the axial position of both H-1 and H-3 also confirmed by ROESY correlation of H-1 with H-3. The NMR data of side chain from C-22 to C-26, was almost superimposable to the data observed in compound 1 indicating the presence of an exomethylene function 25(27). The C-22 configuration of 3a was assigned as α-configuration and was derived by the ROESY experiment that showed key correlations between H-20 (δ_H 2.18) and the protons H-23a (δ_H 1.90)/H-23b (δ_H 1.86) [3] and on the basis of the downfield shift of H-16 at δ_H 4.57 [9].

The linkage of the sugar to the C-26 hydroxyl group was shown by the HMBC correlation between the anomeric carbon at δ_C 103.0 and the two protons at C-26 (δ_H 4.34 and 4.11). Acidic hydrolysis of 3a afforded D-glucose, which was confirmed by GC analysis. Thus compound 3a was established as 26-O-β-D-glucopyranosyl-furosta-5,25(27)diene-1β,3β,22α,26-tetrol 1-O-sulphate.

The spectral data of glycoside 3b indicated its isomeric relationship with sulphated glycoside 3a. In fact, 3b has the same molecular formula determined by HRESI-MS (See Experimental), and ¹H- and ¹³C-NMR spectra (Table 3) almost identical to those of 3a, differing only in the resonances of the carbon atom C-22 (see Table 3). This, in agreement with previous findings [11], indicated that 3b had the opposite configuration at the hemiacetal carbon 22 (22β−ΟΗ) also supported by the H-16 resonance at δ_H 4.38.

COSY, HSQC and HMBC experiments showed that 3b was substituted at its C-1 position by a sulphate group and at C-26 by a β-D-glucopyranosyl moiety, thus compound 3b was defined as: 26-O-β-D-glucopyranosyl-furosta-5,25(27)diene-1β,3β,22β,26-tetrol 1-O-sulphate.

The HRESI-MS spectrum of 4 exhibited a pseudomolecular ion peak at m/z 689.3190 [M−H]⁻ (calcd. for C₃₃H₅₃O₁₃S, 689.3207) indicating the molecular formula C₃₃H₅₃NaO₁₃S in accordance with ¹³C NMR data. The ¹H- and ¹³C-NMR data of the aglycone portion of compound 4, in comparison to those of aglycone of 3a, clearly suggested that 4 differs from 3 by the replacement of the exomethylene group with a secondary methyl group at C-27 (δ_H 0.95, δ_C 17.1) (Table 3). The absolute configuration of C-25 was deduced to be R based on the difference of chemical shifts (Δ_ab = δ_A − δ_B) of the geminal protons H₂-26 (Δ_ab = 0.35 ppm). It has been described that Δ_ab is usually ≥0.57 ppm in 25S compounds and ≤0.48 ppm in 25R compounds [12].

The presence of the sulphate group was confirmed after solvolysis in a dioxane-pyridine mixture that afforded a less polar desulphated derivative 4a, which gave a pseudomolecular ion at m/z 615 [M+Na]⁺. The analysis of NMR spectra showed a high field shift of H-1 at δ_H 3.34 (vs. δ_H 4.01) and C-1 at δ_C 78.6 (vs. δ_C 85.6), confirming the location of the sulphate at C-1. A moderate upfield shift was observed also for the CH₃-19 at δ_H 1.05 (vs. δ_H 1.10 in the natural compound). The solvolysis reaction led to the loss of a H₂O molecule as determined by ESI-MS data and by appearance in the ¹H-NMR spectrum of one allylic methyl group at δ_H 1.60 assigned to C-21.

The NMR data (COSY, TOCSY, HSQC, HMBC) for the sugar portion, were superimposable with those of compound 3a and 3b also confirmed by acidic hydrolysis and GC sugar analysis. Thus compound 4 was elucidated as (25R),26-O-β-D-glucopyranosyl-furost-5-ene-1β,3β,22α,26-tetrol 1-O-sulphate.

In previous studies on the crude extracts from the rhizome of Ruscus aculeatus L., Oulad-Ali et al. [7] reported the isolation of a compound constitutionally identical to compound 4. The stereochemistry at C-22 was left unassigned. Comparison between the ¹³C-NMR data of the two compounds evidenced some small but not insignificant differences, pointing to a stereoisomeric relationship.

Five known compounds were additionally isolated, namely ceparoside A (5) [13]; ruscoponticoside E (6) [14]; ceparoside B (7) [13]; 26-O-β-D-glucopyranosyl-furosta-5,20(22), 25(27)-triene-1β,3β,26-triol 1-O-[α-L-rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside] (8) [15]; and spirosta-5,25(27)-diene-1β,3β-diol 1-O-[α-L-rhamnopyranosyl-(1→2)-O-α-L-arabinopyranoside] (9) [15].

Besides saponins and furostanol glycosides, the hexane extract of the rhizome contains also several minor sterols (campesterol, stigmasterol and sitosterol). The identification has been performed by means of MS spectra and NMR data and comparison with literature data. A previous study on sterol composition of Ruscus aculeatus L. was reported by Dunouau et al. [7].

High-resolution ESI mass spectrometry (HRESI-MS) was recorded on a Micromass QTOF spectrometer and electrospray ionization mass spectrometry (ESI-MS) experiments were performed on an Applied Biosystem API 2000 triple-quadrupole mass spectrometer. Optical rotations were determined on a Jasko P-2000 polarimeter. NMR spectra were obtained on a Varian Inova 500 NMR spectrometer (¹H at 500 MHz and ¹³C at 125 MHz) equipped with a Sun hardware, δ (ppm), J in Hz, using solvent signal for calibration (¹³CD₃OD at δ_C 49.0 and residual CD₂HOD at δ_H = 3.31). The Heteronuclear Single-Quantum Coherence (HSQC) spectra were optimized for an average ¹J_CH of 140 Hz; the gradient-enhanced Heteronuclear Multiple Bond Correlation (HMBC) experiment were optimized for a ³J_CH of 8 Hz.

HPLC was performed using a Waters 510 pump equipped with a Rheodyne 7125 injector and a Waters 401 differential refractometer as detector, using a Nucleodur 100-5 C₁₈ column (5 µm, 4.6 mm i.d. × 250 mm); flow rate was 1 mL min⁻¹. Droplet counter-current chromatography (DCCC) was performed on a DCC-A apparatus (Tokyo Rikakikai Co., Tokyo, Japan) equipped with 250 glass-columns.

The GC/MS analysis was carried out with an Agilent Technologies 6890N Network gas chromatograph coupled to an Agilent Technologies 5973 Network quadrupole mass selective spectrometer and provided with a split/splitless injection port. Helium was used as carrier gas at a linear velocity of 40 cm/s. Separation of compounds was performed on a HP-5 MS capillary column (30 m × 0.25 mm, 0.25 µm film thickness, Agilent USA). GC oven temperature was kept constant at 180 °C. The injector temperature was 230 °C. The temperature of the ion source and the transfer line was 250 and 280 °C, respectively. Mass spectra were taken at 70 eV and the mass range was from 40 to 350 amu.

Selected samples of wild growing plants Ruscus aculeatus L. (Ruscaceae) were collected in May of 2009 in the mountain area of the Tuscany region in Italy. Plants were identified at the Dipartimento di Bioscienze e Territorio, (University of Molise) and a voucher specimen is deposited under No. PGT-58-09 in the Herbarium of University of Molise (Pesche, Isernia). Rhizomes were kept frozen at −20 °C until analyzed.

Underground fresh parts (243 g) were semi-thawed, cut and extracted with MeOH (3 × 700 mL) at room temperature. The combined extracts (56 g) were concentrated and subjected to a modified Kupchan’s [16] partitioning procedure as follows. The MeOH extract was dissolved in 10% aqueous methanol and partitioned against n-hexane to furnish a n-hexane extract (483.8 mg). The water content (% v/v) of the MeOH extract was adjusted to 40% and partitioned against CHCl₃, to furnish a CHCl₃ extract (3.74 g). The aqueous phase was concentrated to remove MeOH and then extracted with n-BuOH yielding 9.0 g of glassy material.

The CHCl₃ extract (1.8 g) was fractionated by DCCC using CHCl₃/MeOH/H₂O (7:13:8) in the ascending mode (the lower phase was the stationary phase), flow rate 8 ml/min; 4 ml fractions were collected. Fractions were monitored by TLC on SiO₂ with CHCl₃/MeOH/H₂O (80:18:2) as eluent and combined on the basis of their similar TLC retention factors. Three major fractions were obtained and then separated by HPLC on a Nucleodur 100-5 C18 column (5 µm, 4.6 mm i.d × 250 mm): fraction 1 was purified with MeOH/H₂O (65:35) as eluent, to afford 6.0 mg of known compound 5; fraction 2 was purified with MeOH/H₂O (7:3) to give 1.9 mg of compound 1, 1.5 mg of compound 2. Fraction 3 yielded known compound 9 (35.3 mg).

The n-BuOH extract (2.0 g) was submitted to DCCC with n-BuOH/Me₂CO/H₂O (3:1:5) in the descending mode (the upper phase was the stationary phase). The obtained fractions were monitored by TLC on Silica gel plates with n-BuOH/OHAc/H₂O (12:3:5) and CHCl₃/MeOH/H₂O (80:18:2) as eluents. Two fractions A and B were obtained and purified by HPLC on a Nucleodur 100-5 C18 column (5 µm, 4.6 mm i.d × 250 mm).

Fraction A (195 mg) was separated with MeOH/H₂O (48:52) as eluent (flow rate 1 mL/min) affording 2 mg of compound 3a, 1.9 mg of compound 3b and 2.6 mg of compound 4.

Fraction B (541 mg) was purified by HPLC with MeOH/H₂O (48:52) as eluent and contained known compounds 6 (28.8 mg), 7 (6.2 mg) and 8 (2.7 mg).

Compound 1: Amorphous solid. [α]²⁵_D −29.7 (c 0.05, MeOH); HRESI-MS m/z 993.4698 [M+Na]⁺ (calcd. for C₄₈H₇₄O₂₀Na, 993.4671). The ¹H- and ¹³C-NMR spectral data are listed in Table 1, Table 2.

Compound 2: Amorphous solid. [α]²⁵_D −7.3 (c 0.15, MeOH); HRESI-MS m/z 1007.4857 [M+Na]⁺ (calcd. for C₄₉H₇₆O₂₀Na, 1007.4828). The ¹H- and ¹³C-NMR spectral data are listed in Table 1, Table 2.

Compound 3a: Amorphous solid. [α]²⁵_D −70.5 (c 0.2, MeOH); HRESI-MS m/z 687.3043 [M−H]⁻ (calcd. for C₃₃H₅₁O₁₃S, 687.3050); ESI-MS (+ve ion) m/z 733 [M+Na]⁺. ESI-MS/MS (+ve ion) m/z 631 [M-NaSO₃+H+Na]⁺. IR νmax (KBr disc)/cm⁻¹ 1245, 1086. The ¹H- and ¹³C-NMR spectral data are listed in Table 3.

Compound 3b: Amorphous solid. [α]²⁵_D −75.3 (c 0.19, MeOH); HRESI-MS m/z 687.3030 [M−H]⁻ (calcd. for C₃₃H₅₁O₁₃S, 687.3050); ESI-MS (+ve ion) m/z 733 [M+Na]⁺. The ¹H- and ¹³C-NMR spectral data are listed in Table 3.

Compound 4: Amorphous solid. [α]²⁵_D −29.2 (c 0.26, MeOH); HRESI-MS m/z 689.3190 [M−H]⁻ (calcd. for C₃₃H₅₃O₁₃S, 689.3207). The ¹H- and ¹³C-NMR spectral data are listed in Table 3.

Compound 5: Amorphous solid. [α]²⁵_D −28.0 (c 0.60, MeOH); HRESI-MS m/z 925.4782 [M+Na]⁺ (calcd. for C₄₅H₇₄O₁₈Na, 925.4773). The ¹H- and ¹³C-NMR spectral data are consistent with the published data [12].

Compound 6: Amorphous solid. [α]²⁵_D −30.0 (c 0.93, MeOH); HRESI-MS m/z 909.4465 [M+Na]⁺ (calcd. for C₄₄H₇₀O₁₈Na, 909.4460). The ¹H- and ¹³C-NMR spectral data are consistent with the published data [13].

Compound 7: Amorphous solid. [α]²⁵_D −29.4 (c 0.07, MeOH); HR-ESI-MS m/z 911.4623 [M+Na]⁺ (calcd. for C₄₄H₇₂O₁₈Na, 911.4616). The ¹H- and ¹³C-NMR spectral data are consistent with the published data [12].

Compound 8: Amorphous solid. [α]²⁵_D −4.63 (c 0.08, MeOH); HRESI-MS m/z 891.4361 [M+Na]⁺ (calcd. for C₄₄H₆₈O₁₇Na, 891.4354). The ¹H- and ¹³C-NMR spectral data are consistent with the published data [14].

Compound 9: Amorphous solid. [α]²⁵_D −64.0 (c 0.69, MeOH); HRESI-MS m/z 729.3832 [M+Na]⁺ (calcd. for C₃₈H₅₈O₁₂Na, 729.3826). The ¹H- and ¹³C-NMR spectral data are consistent with the published data [14].

A solution of compound 4 (2.6 mg, 0.0036 mmol) in pyridine (0.5 mL) and dioxane (0.5 mL) was heated at 150 °C for 2 h in a stoppered reaction vial. After the solution was cooled, the mixture was evaporated to dryness and then purified by HPLC on a Nucleodur 100-5 C18 column (5 µm, 4.6 mm i.d. × 250 mm) with MeOH/H₂O 8:2, to give 1.7 mg of desulphated compound 4a. Compound 4a: [α]²⁵_D −7.8 (c 0.17, MeOH); ESI-MS: 615 [M+Na]⁺; selected ¹H-NMR (CD₃OD, 500 MHz) data for compound 4a: 5.55 (1H, br d, 5.4 Hz, H-6), 4.72 (1H, m, H-16), 3.70 (1H, dd, 9.4, 6.5 Hz, H-26a), 3.39 (1H, ovl, H-26b), 3.39 (1H, ovl, H-3), 3.34 (1H, ovl, H-1), 1.60 (3H, s, H₃-21), 1.05 (3H, s, H₃-19), 0.95 (3H, d, 6.6 Hz, H₃-27), 0.72 (3H, s, H₃-18).

A solution of compounds 1–2 (0.5 mg) in anhydrous 2 N HCl-MeOH (0.5 mL) was heated at 80 °C in a stoppered reaction vial. After 2 h, the reaction mixture was cooled, neutralized with Ag₂CO₃, and centrifuged, and the supernatant was taken to dryness under N₂. 1-(Trimethylsilyl)imidazole in pyridine was added and left at room temperature for 15 min. The derivatives were analyzed by GC-MS (HP-5MS capillary column, helium carrier, flow 10 mL min⁻¹ oven temperature 150 °C). GC-MS peaks in the sylilated saponin hydrolysate coeluted with those in silylated standards (methyl rhamnosides, methyl arabinosides and methyl glucosides).