Novel steroidal components from the underground parts of Ruscus aculeatus L.

Article abstract of DOI:10.3390/molecules171214002

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

Full text of DOI:10.3390/molecules171214002

Molecules 2012, 17, 14002-14014; doi:10.3390/molecules171214002
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Steroidal Components from the Underground Parts of
Ruscus aculeatus L.
1
,
1
1
2
Simona De Marino *, Carmen Festa , Franco Zollo and Maria Iorizzi
1
Dipartimento di Chimica delle Sostanze Naturali, Università degli Studi di Napoli “Federico II”,
Via D. Montesano 49, I-80131 Napoli, Italy
2
Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Contrada Fonte Lappone,
I-86090 Pesche (Isernia), Italy
*
Author to whom correspondence should be addressed; E-Mail: sidemari@unina.it;
Tel.: +39-081-678124; Fax: +39-081-678552.
Received: 8 October 2012; in revised form: 16 November 2012 / Accepted: 18 November 2012 /
Published: 26 November 2012
Abstract: 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.
Keywords: Ruscaceae; Ruscus aculeatus L.; furostanol saponins; sulphated steroidal
glycosides; NMR spectroscopy
1
. 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

Molecules 2012, 17
14003
were isolated from its methanolic extract along with four known furostanol and one spirostanol
glycosides 5–9 (Figures 1 and 2).
Figure 1. New compounds isolated from Ruscus aculeatus L.
R1

-L-ara
O
21
21
20
OR
R
AcO
AcO
20
25
25
O
22
22
16
NaO
HO
3
SO
16
O
O
1'
O
1'''
O
O
O
O
1
9
14
HO
1
9
14
HO
1'
OH
-D-glc
OH
3
3
1''
O
HO
-D-glc
HO HO
HO
HO
HO
HO
OH
-L-rha
OH

R1= CH
2
3
3
4
a
R=
R=
R=
1
2
R=H
OH
OH
R=CH3
R = CH
2
b
1
R =(25R)-CH
1
3
Figure 2. Known compounds isolated from Ruscus aculeatus L.
R
1

-L-ara
O
R
HO
HO
OCH3
OH
O
O
5
6
7
R=
R=
R=
R =(25R)-CH3
1
O
O
O
HO
OH
1
R =CH2
OH
HO
O
HO
R =(25R)-CH3
HO

1
-
D-glc
HO
HO
OH
-L-rha


-L-ara
HO
HO
HO -L-ara
O
O
O
O
O
HO
O
O
O
O
O
O
HO
OH
-D-glc
O
O
HO
HO HO
HO
HO
HO
HO
HO
OH
-L-rha
OH
-L-rha

8
9
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.

Molecules 2012, 17
. Results and Discussion
Structure Analysis and Characterization of Compounds 1–4
14004
2
Compound 1 was obtained as a white amorphous solid. The molecular formula was determined to
+
be C H O from the molecular ion peak [M+Na] at m/z 993.4698 (calcd. for C H O Na,
4
8
74 20
48 74 20
1
9
93.4671) in the positive HRESI-MS. The analysis of the H-NMR spectrum of 1, in combination with
HSQC data, showed signals for three anomeric protons 
H
4.95/
.28/ 103.1, suggesting the presence of three sugar moieties.
C
101.7, 
H
4.50/
C
100.1 and 
H
4
C
1
In addition, the H-NMR spectrum (Table 1) showed signals for two tertiary methyl groups at 
.09 and 0.86 (each 3H, s) and one secondary methyl group at 
H
1
H
1.02 (3H, d, 6.8 Hz), as well as one
signal for a trisubstituted double bond ( 5.55, 1H, br d, 5.6 Hz) and for an exometylene group
H
(

H
H
5.09 and 4.92 each 1H, br s), three methine proton signals at  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  4.33 and 4.13 (each 1H, d, 12.5 Hz) ascribable to a primary alcoholic function.
H
1
3
The C-NMR spectrum showed three secondary alcoholic functions at 
C
83.9, 81.8 and 68.6, one
111.7, suggesting the
primary alcoholic function at  72.4 and a hemiacetalic carbon signal at 
C
C
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 (
assigned on the basis of key ROESY correlations between H-20 (
1.84) and of the downfield shift of H-16 at  4.56 [9].
C
83.9) and C-26 ( 72.4). The C-22 -configuration of 1 was
C
H
2
2.14) and the protons H -23
(

H
H
1
As concerning the sugar portion, in addition to the carbinol protons, the H-NMR spectrum (Table
2
) showed signals at 
groups [ 170.8 and 170.9 (C=O); 
indicative of a 6-deoxyhexopyranose unit (Table 2).
H
2.03 and 2.04 (each 3H, s), ascribable to the methyl groups of two acetyl
C
C
20.7 and 20.8 (CH )], and one signal at  1.26 (3H, d, 6.3 Hz)
3
H
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 JH1-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 JH1-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
H
5.05) and H-4′ ( 5.30) and for the upfield shift of C-2′
(

C
73.5) and C-5′ ( 64.5) in comparison with the data reported for the authentic sample,
C
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  5.05 (H-3′) and
H

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′′′

Molecules 2012, 17
14005
of Glc at  4.28 and C-26 at  72.4, implied that the glucose unit is attached to C-26 of the aglycone,
H
C
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 ( 4.50) and C-1 at
C 83.9. Furthermore the anomeric proton of rhamnose at  4.95 was correlated with C-2′ of arabinose
H
H
C
at  73.5 which supported the proposed sequence of the disaccharidic chain linked at C-1 of
the aglycone.
Figure 3. Key HMBC correlations for compound 1.
O
2
1
OH
O
O
1
8
26
4'
O
2
2
2
'
O
17
1
'
19
13
14
O
3
'
O
1'''
9
O
O
1
HO
O
10
OH
1
''
5
O
HO
HO HO
HO
HO
OH
Thus the structure of compound 1 was established as 26-O--D-glucopyranosylfurosta-
5
,25(27)-diene-1,3,22,26-tetrol
arabino- pyranoside].
HRESI-MS of compound 2 showed a pseudomolecular ion peak at m/z 1007.4857 [M+Na]
1-O-[-L-rhamnopyranosyl-(1′′→2′)-O-(3′,4′-di-O-acetyl)--L-
+
corresponding to the molecular formula C49
H
76
O
20, which differs from that of compound 1 only in the
1
13
gain of 14 u.m.a. H-NMR [
H
3.16 (3H, s)] and C-NMR [
C
113.3 (C-22) and 47.5 (-OCH
3
)] 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  3.16 and C-22 ( 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  3.16 and the H-16 at  4.38 and between H-20 ( 2.22) and H-23a ( 1.90)/H-23b
1.84) indicating an -orientation of the methoxy group [3].
Analysis of COSY, HSQC and HMBC experiments revealed that 2 possessed sugar moieties
H
C
H
H
H
H
(
H
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--D-glucopyranosyl-22-methoxy-furosta-5,25(27)diene-1,3,26-triol 1-O-[-L-rhamnopyranosyl-
(
1′′→2′)-O-(3′,4′-di-O-acetyl)--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 C33
H
51
O
13S, 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−NaSO
3
+H+Na] in the ESI-MS/MS spectrum recorded in a positive ion mode,
−
1
corresponding to the loss of a SO
3
Na from the parent ion, and by IR bands at 1245 and 1086 cm .

Molecules 2012, 17
14006
1
13
Table 1. H- and C-NMR data (CD OD, 500 and 125 MHz) data of the aglycon portions
3
of compounds 1 and 2.
1
2
Position
a
a
δ
H
δ
C
δ
H
δ
C
1
3.38 m
2.10 m
1.69 m
3.33 m
2.23 ovl
-
83.9
6.8
3.44 dd (11.8, 3.7)
2.10 m
1.69 m
3.35 m
2.21 ovl
-
83.7
2
2
a
3
37.0
b
3
68.6
43.0
69.0
43.2
4
5
6
139.2
125.4
139.2
125.6
5.55 br d (5.6)
1.97 ovl
1.53 ovl
1.54 ovl
1.25 ovl
-
5.56 br d (5.4)
1.96 ovl
1.53 ovl
1.55 ovl
1.25 ovl
-
7
a
3
2.3
32.6
7
b
8
33.6
50.9
43.5
33.8
51.1
43.4
9
1
0
1
1
1
1
1a
1b
2a
2b
2.55 m
1.49 m
1.68 ovl
1.21 m
-
2.56 m
1.48 m
1.70 ovl
1.21 m
-
2
4
4.7
0.7
25.0
41.1
1
3
4
41.6
57.2
41.4
57.5
1
1.13 m
1.98 ovl
1.29 m
4.56 m
1.77 m
0.86 s
1.13 m
1.97 ovl
1.26 ovl
4.38 m
1.73 ovl
0.86 s
1
5a
5b
3
2.5
32.6
1
1
6
7
8
9
0
1
2
81.8
63.7
16.7
14.9
40.4
15.4
111.7
82.4
65.0
17.1
15.1
41.0
15.9
113.3
1
1
1
2
2
2
1.09 s
1.12 s
2.14 m
1.02 d (6.8)
-
2.22 ovl
1.04 d (6.8)
-
2
3a
3b
1.90 m
1.84 m
2.18 ovl
-
1
.84 m
37.4
32.1
2
2
4
2.29 ovl
-
28.3
28.5
2
5
147.4
147.1
2
2
2
2
6a
6b
7a
7b
4.33 d (12.5)
4.13 d (12.5)
5.09 br s
4.92 br s
4.34 d (12.6)
4.13 d (12.6)
5.08 br s
4.93 br s
3.16 s
7
2.4
72.4
1
12.1
112.2
47.5
-
OCH3
a
1
13
Ovl: overlapped signals; Coupling constants are in parentheses and given in Hertz. H- and C-NMR
assignments aided by COSY, HSQC and HMBC experiments.

Molecules 2012, 17
14007
1
13
Table 2. H- and C-NMR data (CD OD, 500 and 125 MHz) data of the sugar portions of
3
compounds 1 and 6.
a
b
1
6
Position
-L-Ara
c
c
δ
H
δ
C
δ
H
δ
C

1
2
3
4
′
′
′
′
4.50 d (7.4)
3.85 ovl
100.1
73.5
75.7
70.6
4.26 d (7.1)
3.70 ovl
100.7
75.4
75.7
70.5
5.05 dd (9.7, 3.1)
5.30 br s
3.64 dd (9.5, 3.2)
3.74 ovl
3
3
.89 ovl
.73 ovl
3.85 dd (12.1, 2.0)
3.48 dd (12.1, 3.0)
5
′
64.5
67.2
3
3
4
4
′-COCH3
′-COCH3
′-COCH3
′-COCH3
2.04 s
20.8
170.9
20.7
2.03 s
170.8

-L-Rha
1
2
3
4
5
6
′′
′′
′′
′′
′′
′′
4.95 br d (1.2)
3.72 ovl
101.7
72.1
71.8
73.8
69.8
18.3
5.29 br d (1.2)
3.88 ovl
101.3
72.0
71.8
73.8
69.5
18.0
3.62 dd (9.6, 3.3)
3.39 t (9.6)
4.08 m
3.69 ovl
3.40 t (9.7)
4.08 m
1.26 d (6.3)
1.26 d (6.2)

-D-Glc (C26)
1
2
3
4
5
′′′
′′′
′′′
′′′
′′′
4.28 d (7.7)
3.22 t (8.3)
3.35 ovl
103.1
74.9
77.9
71.5
77.7
4.28 d (7.6)
3.21 t (8.4)
3.35 ovl
103.0
74.9
77.9
71.4
77.6
3.27 ovl
3.28 ovl
3.25 ovl
3.26 ovl
3
.87 ovl
3.87 ovl
6
′′′
62.6
62.6
3
.66 dd (12.1, 4.5)
3.67 dd (12.0, 4.5)
a
b
The chemical shift values of the sugar portion of 2 are identical to those reported for 1; Data reported for
the authentic sample, ruscoponticoside E (6), also isolated in the present study. Ovl: overlapped signals;
c
1
13
Coupling constants are in parentheses and given in Hertz. H and C assignments aided by COSY, TOCSY,
HSQC and HMBC experiments.
1
Preliminary H-NMR analysis of 3a (Table 3) indicated the steroid glycoside nature of the
1
compound. The H-NMR spectrum showed three methyl signals: two tertiary (
H
0.86 and 1.10) and
4.28. Its C-NMR spectrum exhibited
3 carbon signals, with 27 being attributable to the aglycone and six attributable to the monosaccharide
1
3
one secondary (
H
1.04), and one anomeric proton signal at 
H
3
1
3
unit. The C-NMR spectrum further showed three secondary alcoholic functions at 
8.6, one primary alcoholic function at  72.6 and a hemiacetalic carbon signal at 
a furostane nature for the steroidal aglycone of 3a.
C
85.5, 81.8 and
6
C
C
111.1 indicating
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.

Molecules 2012, 17
14008
1
13
Table 3. H- and C-NMR (CD OD, 500 and 125 MHz) data of compounds 3a, 3b and 4.
3
3
a
3b
4
Position
a
a
a

H

C

H

C

H

C
1
4.03 dd (11.9, 3.8)
2.55 m
85.5
4.02 dd (11.9, 3.8)
2.56 m
85.4
4.01 dd (11.8, 4.0)
2.55 m
85.6
2
2
a
b
3
8.8
38.7
38.8
1.69 m
1.69 m
1.67 m
3
.43 dddd
3.41 dddd
(12.0, 12.0, 6.4, 6.4)
2.23 ovl
-
3.45 dddd
(12.1, 12.1, 6.5, 6.5)
2.21 m (2H)
-
3
68.6
68.6
68.7
(
12.0, 12.0, 6.4, 6.4)
2.21 ovl
-
5.61 br d (5.2)
1.98 ovl
1.55 ovl
1.55 ovl
1.37 ovl
-
4
5
6
43.2
138.7
126.5
43.2
139.0
126.4
43.0
138.8
126.5
5.60 br d (5.2)
1.99 ovl
1.57 ovl
1.56 ovl
1.37 ovl
-
5.60 br d (5.4)
1.96 m
7
a
3
2.8
33.0
32.6
7
b
1.54 ovl
1.54 ovl
1.35 ovl
-
8
9
33.8
50.6
43.7
33.8
50.7
43.6
33.8
50.7
43.7
1
0
1
1
1
1
1
1
1a
1b
2a
2b
3
4
2.36 br d (12.6)
1.52 ovl
1.71 ovl
1.26 m
2.35 br d (12.6)
1.52 ovl
1.70 ovl
1.28 m
2.36 br d (12.4)
1.48 ovl
1.70 ovl
1.24 ovl
-
2
4
4.0
0.8
24.0
40.9
24.0
40.9
-
41.2
57.4
-
41.4
57.5
41.4
57.5
1.17 m
1.17 m
1.18 m
1
1
1
1
1
1
2
2
2
5a
5b
1.99 ovl
1.32 ovl
4.57 m
1.78 ovl
0.86 s
2.00 ovl
1.33 ovl
4.38 m
1.74 ovl
0.87 s
1.95 ovl,
1.26 ovl
4.57 q (5.6)
1.71 ovl
0.83 s
3
2.7
32.6
32.7
6
7
8
9
0
1
2
81.8
64.0
16.8
14.5
40.8
15.8
111.1
82.2
64.1
16.9
14.5
40.9
16.0
113.7
82.0
64.1
16.9
14.6
41.1
16.1
111.3
1.10 s
1.10 s
1.10 s
2.18 ovl
1.04 d (6.7)
-
2.19 ovl
1.01 d (6.7)
-
2.16 ovl
0.99 d (7.1)
-
2
2
2
2
3a
3b
4a
4b
1.90 ovl
1.86 ovl
2.22 ovl
2.15 ovl
-
1.91 ovl
1.85 ovl
2.21 ovl
2.15 ovl
-
3
2
7.5
8.5
37.3
1.61 ovl
31.4
1.57 ovl
1.12 m
1.72 m
28.4
147.3
72.4
28.6
34.8
75.6
2
5
147.0
2.6
26a
26b
27a
27b
4.34 d (12.5)
4.11 d (12.5)
5.09 br s
4.93 br s
4.33 d (12.4)
4.12 d (12.4)
5.10 br s
4.94 br s
3.74 dd (9.4, 6.5)
3.39 ovl
7
1
11.9
111.9
0.95 d (6.6)
17.1
Glucose
1
2
3
4
5
′
′
′
′
′
4.28 d (7.8)
3.22 t (8.5)
3.36 ovl
3.28 ovl
3.27 ovl
103.0
74.9
77.8
71.5
77.7
4.27 d (7.8)
3.20 t (8.5)
3.34 ovl
3.28 ovl
3.26 ovl
103.1
75.0
77.8
71.4
77.8
4.24 d (7.7)
3.19 t (8.4)
3.35 ovl
3.27 ovl
3.26 ovl
104.2
74.8
77.9
71.5
77.7
3
.86 d (11.7)
3.88 d (11.7)
3.65 dd (11.7, 5.2)
3.87 d (11.9)
3.68 dd (11.9, 5.1)
6
′
62.6
62.6
62.7
3
.66 dd (11.7, 5.2)
a
1
13
Ovl: overlapped signals; Coupling constants are in parentheses and given in Hertz. H- and C- assignments
aided by COSY, TOCSY, HSQC and HMBC experiments.

Molecules 2012, 17
14009
The location of the sulphate group at position-1 was inferred by the downfield shift of the
corresponding nuclei (H-1,  4.03 and C-1,  85.5) [7]. The relative stereochemistry at C-1 and C-3
H
C
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 ( 2.18) and
H
the protons H-23a ( 1.90)/H-23b ( 1.86) [3] and on the basis of the downfield shift of H-16 at
H
H

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  103.0 and the two protons at C-26 ( 4.34 and 4.11). Acidic hydrolysis of
C
H
3
a afforded D-glucose, which was confirmed by GC analysis. Thus compound 3a was established as
26-O--D-glucopyranosyl-furosta-5,25(27)diene-1,3,22,26-tetrol 1-O-sulphate.
The spectral data of glycoside 3b indicated its isomeric relationship with sulphated glycoside 3a. In
1
fact, 3b has the same molecular formula determined by HRESI-MS (See Experimental), and H- and
1
3
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  4.38.
H
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:
2
6-O--D-glucopyranosyl-furosta-5,25(27)diene-1,3,22,26-tetrol 1-O-sulphate.
The HRESI-MS spectrum of 4 exhibited a pseudomolecular ion peak at m/z 689.3190 [M−H]
−
(
1
calcd. for C33
H
53
O
13S, 689.3207) indicating the molecular formula C33
H
53NaO13S in accordance with
3
1
13
C NMR data. The H- and C-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 ( 0.95,  17.1) (Table 3). The absolute
configuration of C-25 was deduced to be R based on the difference of chemical shifts ( =  −  )
H
C
ab
A
B
of the geminal protons H
5S compounds and 0.48 ppm in 25R compounds [12].
The presence of the sulphate group was confirmed after solvolysis in a dioxane-pyridine mixture
2
-26 (ab = 0.35 ppm). It has been described that ab is usually 0.57 ppm in
2
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  3.34 (vs.  4.01) and
H
H
C-1 at 
was observed also for the CH
C
78.6 (vs. 
C
85.6), confirming the location of the sulphate at C-1. A moderate upfield shift
-19 at  1.05 (vs.  1.10 in the natural compound). The solvolysis
O molecule as determined by ESI-MS data and by appearance in the
1.60 assigned to C-21.
3
H
H
reaction led to the loss of a H
2
1
H-NMR spectrum of one allylic methyl group at 
H
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

Molecules 2012, 17
14010
compound 4 was elucidated as (25R),26-O--D-glucopyranosyl-furost-5-ene-1,3,22,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
13
C-22 was left unassigned. Comparison between the 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) [13]; 26-O--D-glucopyranosyl-furosta-5,20(22), 25(27)-triene-1,3,26-
triol 1-O-[-L-rhamnopyranosyl-(1→2)-O--L-arabinopyranoside] (8) [15]; and spirosta-5,25(27)-
diene-1,3-diol 1-O-[-L-rhamnopyranosyl-(1→2)-O--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].
3. Experimental
3.1. 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
1
13
spectrometer ( H at 500 MHz and C at 125 MHz) equipped with a Sun hardware,  (ppm), J in Hz,
1
3
using solvent signal for calibration ( CD
3
OD at δ
C
49.0 and residual CD
2
HOD at δ
H
1
= 3.31). The
Heteronuclear Single-Quantum Coherence (HSQC) spectra were optimized for an average JCH of 140 Hz;
the gradient-enhanced Heteronuclear Multiple Bond Correlation (HMBC) experiment were optimized
3
for a JCH 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
−1
i.d. × 250 mm); flow rate was 1 mL min . 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
4
0
0 cm/s. Separation of compounds was performed on a HP-5 MS capillary column (30 m × 0.25 mm,
.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.

Molecules 2012, 17
14011
3
.2. Plant Material
Selected samples of wild growing plants Ruscus aculeatus L. (Ruscaceae) were collected in May of
2
009 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.
3
.3. 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 , to furnish a CHCl3
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.
The CHCl
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 SiO with CHCl /MeOH/H O (80:18:2) as eluent and
3 3 2
extract (1.8 g) was fractionated by DCCC using CHCl /MeOH/H O (7:13:8) in the
2
3
2
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/H
2
O (65:35) as eluent, to afford 6.0 mg of known compound 5; fraction 2
was purified with MeOH/H
2
O (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).
2 2
The n-BuOH extract (2.0 g) was submitted to DCCC with n-BuOH/Me CO/H O (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/H O (12:3:5) and CHCl /MeOH/H O (80:18:2) as
2
3
2
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).
2
Fraction A (195 mg) was separated with MeOH/H O (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/H
2
O (48:52) as eluent and contained
known compounds 6 (28.8 mg), 7 (6.2 mg) and 8 (2.7 mg).
2
D
5
+
Compound 1: Amorphous solid. [α]
−29.7 (c 0.05, MeOH); HRESI-MS m/z 993.4698 [M+Na]
1
13
(
calcd. for C48 20Na, 993.4671). The H- and C-NMR spectral data are listed in Tables 1–2.
74
H O
2
D
5
+
Compound 2: Amorphous solid. [α]
−7.3 (c 0.15, MeOH); HRESI-MS m/z 1007.4857 [M+Na]
1
13
(
calcd. for C49 20Na, 1007.4828). The H- and C-NMR spectral data are listed in Tables 1–2.
76
H O
2
D
5
−
Compound 3a: Amorphous solid. [α]
−70.5 (c 0.2, MeOH); HRESI-MS m/z 687.3043 [M−H]
+
(
calcd. for C H O S, 687.3050); ESI-MS (+ve ion) m/z 733 [M+Na] . ESI-MS/MS (+ve ion) m/z
3
3
51 13

Molecules 2012, 17
14012
+
−1
1
13
6
31 [M-NaSO +H+Na] . IR max (KBr disc)/cm 1245, 1086. The H- and C-NMR spectral data
3
are listed in Table 3.
2
D
5
−
Compound 3b: Amorphous solid. [α]
−75.3 (c 0.19, MeOH); HRESI-MS m/z 687.3030 [M−H]
+
1
13
(
calcd. for C H O S, 687.3050); ESI-MS (+ve ion) m/z 733 [M+Na] . The H- and C-NMR
33 51 13
spectral data are listed in Table 3.
2
D
5
−
Compound 4: Amorphous solid. [α]
−29.2 (c 0.26, MeOH); HRESI-MS m/z 689.3190 [M−H]
1
13
(
calcd. for C H O S, 689.3207). The H- and C-NMR spectral data are listed in Table 3.
3
3
53 13
2
D
5
+
Compound 5: Amorphous solid. [α]
calcd. for C45
−28.0 (c 0.60, MeOH); HRESI-MS m/z 925.4782 [M+Na]
1
13
(
H
74
O
18Na, 925.4773). The H- and C-NMR spectral data are consistent with the
published data [12].
2
D
5
+
Compound 6: Amorphous solid. [α]
−30.0 (c 0.93, MeOH); HRESI-MS m/z 909.4465 [M+Na]
1
13
(
70
calcd. for C44H O18Na, 909.4460). The H- and C-NMR spectral data are consistent with the
published data [13].
2
D
5
+
Compound 7: Amorphous solid. [α]
−29.4 (c 0.07, MeOH); HR-ESI-MS m/z 911.4623 [M+Na]
1
13
(
72
calcd. for C44H O18Na, 911.4616). The H- and C-NMR spectral data are consistent with the
published data [12].
2
D
5
+
Compound 8: Amorphous solid. [α]
−4.63 (c 0.08, MeOH); HRESI-MS m/z 891.4361 [M+Na]
1
13
(
68
calcd. for C44H O17Na, 891.4354). The H- and C-NMR spectral data are consistent with the
published data [14].
2
D
5
+
Compound 9: Amorphous solid. [α]
−64.0 (c 0.69, MeOH); HRESI-MS m/z 729.3832 [M+Na]
1
13
(
calcd. for C38
58
H O12Na, 729.3826). The H- and C-NMR spectral data are consistent with the
published data [14].
3
.4. 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 (5 µm, 4.6 mm
2
i.d. × 250 mm) with MeOH/H O 8:2, to give 1.7 mg of desulphated compound 4a. Compound 4a:
2
5
+
1
[
] −7.8 (c 0.17, MeOH); ESI-MS: 615 [M+Na] ; selected H-NMR (CD
3
OD, 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),
.39 (1H, ovl, H-26b), 3.39 (1H, ovl, H-3), 3.34 (1H, ovl, H-1), 1.60 (3H, s, H -21), 1.05 (3H, s,
-19), 0.95 (3H, d, 6.6 Hz, H -27), 0.72 (3H, s, H -18).
D
3
3
H
3
3
3
3
.5. 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 CO , and
2
3

Molecules 2012, 17
14013
centrifuged, and the supernatant was taken to dryness under N . 1-(Trimethylsilyl)imidazole in
2
pyridine was added and left at room temperature for 15 min. The derivatives were analyzed by GC-MS
−1
(
HP-5MS capillary column, helium carrier, flow 10 mL min 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).
4. 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.
Acknowledgments
MS and NMR spectra were provided by Centro di Servizio Interdipartimentale di Analisi
Strumentale (CSIAS), Università di Napoli “Federico II”, Napoli, Italy.
Conflict of Interest
The authors declare no conflict of interest.
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©
2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
http://creativecommons.org/licenses/by/3.0/).
(