Novel Steroidal Components from the Underground Parts of Ruscus aculeatus L

Two new furostanol saponins 1–2 and three new sulphated glycosides 3a,b and 4 were isolated from the underground parts of Ruscus aculeatus L., along with four known furostanol and one spirostanol saponins 5–9 and three free sterols. All of the structures have been elucidated on the basis of spectroscopic data 1D and 2D NMR experiments, MS spectra and GC analyses.


Introduction
Ruscus aculeatus L. (Ruscaceae family) is a small evergreen shrub and a widely distributed European plant. The hydroalcoholic extract of its rhizome is commonly used as a vascular preventive and vascular tonic in pharmaceutical preparations [1]. Previous chemical analysis of secondary metabolites have been described in R. aculeatus L. [2,3], R. hypoglossum L. [4], R. colchicus Y. Yeo [5] and R. ponticus Wor. [6]. The steroidal constituents of this herb, including spirostane, furostane and triterpene type, are the main secondary metabolites isolated from the rhizome and leaves [7].
In the present work we performed a phytochemical investigation of the fresh underground parts of Ruscus aculeatus L. Two new furostanol saponins 1-2 and three new sulphated glycosides 3a,b and 4 OPEN ACCESS were isolated from its methanolic extract along with four known furostanol and one spirostanol glycosides 5-9 (Figures 1 and 2). The composition of free sterols was also determined and campesterol, stigmasterol, sitosterol were the major components. All new components are bisdesmosidic saponins with a diglycoside moiety linked at C-1 and a glucose unit linked at C-26. The isolation of sulphated compounds in Ruscus aculeatus L. are previously reported only by Oulad-Ali et al. [8].
Their structures were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRESI-MS, and chemical methods. Herein, we report the isolation and structural elucidation of the new compounds 1-4. The 13 C-NMR spectrum showed three secondary alcoholic functions at  C 83.9, 81.8 and 68.6, one primary alcoholic function at  C 72.4 and a hemiacetalic carbon signal at  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 ( C 83.9) and C-26 ( C 72.4). The C-22 -configuration of 1 was assigned on the basis of key ROESY correlations between H-20 ( H 2.14) and the protons H 2 -23 ( H 1.84) and of the downfield shift of H-16 at  H 4.56 [9].

Structure Analysis and Characterization of Compounds
As concerning the sugar portion, in addition to the carbinol protons, the 1 H-NMR spectrum ( Table  2) showed signals at  H 2.03 and 2.04 (each 3H, s), ascribable to the methyl groups of two acetyl groups [ C 170.8 and 170.9 (C=O);  C 20.7 and 20.8 (CH 3 )], and one signal at  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 -glucopyranosyl (Glc), one -arabinopyranosyl (Ara) and one -rhamnopyranosyl (Rha) units. The relatively large J H1-H2 values (7.4-8.0 Hz) indicated a -orientation for the anomeric center of glucose and an -orientation for that of arabinose in their pyranose form, whereas a small J H1-H2 coupling (1.2 Hz) indicated the -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′ ( H 5.05) and H-4′ ( H 5.30) and for the upfield shift of C-2′ ( C 73.5) and C-5′ ( 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  H 5.05 (H-3′) and  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  H 4.28 and C-26 at  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 ( H 4.50) and C-1 at  C 83.9. Furthermore the anomeric proton of rhamnose at  H 4.95 was correlated with C-2′ of arabinose at  C 73.5 which supported the proposed sequence of the disaccharidic chain linked at C-1 of the aglycone.  (Table 1).
A HMBC experiment confirmed this hypothesis and showed a correlation between the methoxy group at  H 3.16 and C-22 ( 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  H 3.16 and the H-16 at  H 4.38 and between H-20 ( H 2.22) and H-23a ( H 1.90)/H-23b ( H 1.84) indicating an -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 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 C 33 H 51 O 13    Preliminary 1 H-NMR analysis of 3a (Table 3) indicated the steroid glycoside nature of the compound. The 1 H-NMR spectrum showed three methyl signals: two tertiary ( H 0.86 and 1.10) and one secondary ( H 1.04), and one anomeric proton signal at  H 4.28. Its 13 C-NMR spectrum exhibited 33 carbon signals, with 27 being attributable to the aglycone and six attributable to the monosaccharide unit. The 13 C-NMR spectrum further showed three secondary alcoholic functions at  C 85.5, 81.8 and 68.6, one primary alcoholic function at  C 72.6 and a hemiacetalic carbon signal at  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,  H 4.03 and C-1,  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 -configuration and was derived by the ROESY experiment that showed key correlations between H-20 ( H 2. 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 1 H-and 13 C-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 (22) also supported by the H-16 resonance at  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 -D-glucopyranosyl moiety, thus compound 3b was defined as:  Table 3). The absolute configuration of C-25 was deduced to be R based on the difference of chemical shifts ( ab =  A −  B ) of the geminal protons H 2 -26 ( ab = 0.35 ppm). It has been described that  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  H 3.34 (vs.  H 4.01) and C-1 at  C 78.6 (vs.  C 85.6), confirming the location of the sulphate at C-1. A moderate upfield shift was observed also for the CH 3 -19 at  H 1.05 (vs.  H 1.10 in the natural compound). The solvolysis reaction led to the loss of a H 2 O molecule as determined by ESI-MS data and by appearance in the 1 H-NMR spectrum of one allylic methyl group at  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 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 13 C-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)  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].

General
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 ( 1 H at 500 MHz and 13 C at 125 MHz) equipped with a Sun hardware,  (ppm), J in Hz, using solvent signal for calibration ( 13 CD 3 OD at δ C 49.0 and residual CD 2 HOD at δ H = 3.31). The Heteronuclear Single-Quantum Coherence (HSQC) spectra were optimized for an average 1 J CH of 140 Hz; the gradient-enhanced Heteronuclear Multiple Bond Correlation (HMBC) experiment were optimized for a 3 J CH of 8 Hz.
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.

Compound Isolation
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 CHCl 3 , to furnish a CHCl 3 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.

Solvolysis of Compound 4 Giving 4a
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 (

Methanolysis of 1-2: Sugar Analysis
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 Ag 2 CO 3 , and centrifuged, and the supernatant was taken to dryness under N 2 . 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).

Conclutions
Two new furostanol saponins 1-2 and three new sulphated glycosides 3a, 3b and 4 were isolated from the underground parts of Ruscus aculeatus L., along with four known furostanol and one spirostanol saponins 5-9 and three free sterols. The new compounds add knowledge in the field of isolation and structural characterization of new metabolites from natural sources.