Steroidal glycosides from Ruscus ponticus

Article abstract of DOI:10.1016/j.phytochem.2011.01.033

A comparative metabolite profiling of the underground parts and leaves of Ruscus ponticus was obtained by an HPLC-ESIMSⁿ method, based on high-performance liquid chromatography coupled to electrospray positive ionization multistage ion trap mass spectrometry. The careful study of HPLC-ESIMSⁿ fragmentation pattern of each chromatographic peak, in particular the identification of diagnostic product ions, allowed us to get a rapid screening of saponins belonging to different classes, such as dehydrated/or not furostanol, spirostanol and pregnane glycosides, and to promptly highlight similarities and differences between the two plant parts. This approach, followed by isolation and structure elucidation by 1D- and 2D-NMR experiments, led to the identification of eleven saponins from the underground parts, of which two dehydrated furostanol glycosides and one new vespertilin derivative, and nine saponins from R. ponticus leaves, never reported previously. The achieved results highlighted a clean prevalence of furostanol glycoside derivatives in R. ponticus leaves rather in the underground parts of the plant, which showed a wider structure variety. In particular, the occurrence of dehydrated furostanol derivatives, for the first time isolated from a Ruscus species, is an unusual finding which makes unique the saponins profile of R. ponticus.

Full text of DOI:10.1016/j.phytochem.2011.01.033

Phytochemistry 72 (2011) 651–661
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Steroidal glycosides from Ruscus ponticus
a
b
a
a
b
Assunta Napolitano , Tamar Muzashvili , Angela Perrone , Cosimo Pizza , Ether Kemertelidze ,
Sonia Piacente
a
a,
⇑
Dipartimento di Scienze Farmaceutiche e Biomediche, Università degli Studi di Salerno, Via Ponte Don Melillo, I-84084 Fisciano, Italy
Institute of Pharmacochemistry, P. Sarajishvili Street 36, 0159 Tbilisi, Georgia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A comparative metabolite profiling of the underground parts and leaves of Ruscus ponticus was obtained by
Received 23 November 2010
Received in revised form 21 January 2011
Available online 26 February 2011
n
an HPLC–ESIMS method, based on high-performance liquid chromatography coupled to electrospray
n
positive ionization multistage ion trap mass spectrometry. The careful study of HPLC–ESIMS fragmenta-
tion pattern of each chromatographic peak, in particular the identification of diagnostic product ions,
allowed us to get a rapid screening of saponins belonging to different classes, such as dehydrated/or not
furostanol, spirostanol and pregnane glycosides, and to promptly highlight similarities and differences
between the two plant parts. This approach, followed by isolation and structure elucidation by 1D- and
Keywords:
Ruscus ponticus
Steroidal glycosides
HPLC–ESIMS comparative profile
2D-NMR experiments, led to the identification of eleven saponins from the underground parts, of which
two dehydrated furostanol glycosides and one vespertilin derivative, and nine saponins from
R. ponticus leaves, never reported previously. The achieved results highlighted a clean prevalence of furost-
anol glycoside derivatives in R. ponticus leaves rather in the underground parts of the plant, which showed a
wider structure variety. In particular, the occurrence of dehydrated furostanol derivatives, for the first time
isolated from a Ruscus species, is an unusual finding which makes unique the saponins profile of R. ponticus.
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
Ruscus ponticus Woronow ex grossh. (Ruscaceae) is an ever-
Recently, we have developed an analytical method, based on
high-performance liquid chromatography coupled to electrospray
positive ionization multistage ion trap mass spectrometry (HPLC–
n
green, perennial, 30–50 cm tall shrub-like relict plant, with fo-
lium-like cladodes and red fruits, widespread in Crimea and
Caucasus, particularly in the forests of West and East Georgia
ESIMS ), as an effective tool to rapidly identify and guide the isola-
tion of target saponins from the leaves of Ruscus colchicus Y. Yeo
(Perrone et al., 2009).
n
(Gagnidze, 2005). R. ponticus is well known in this country for
This HPLC–ESIMS method allowed to define the mass fragmen-
the preparation of ruscoponin, obtained from the underground
parts of the plant. Some pharmacological properties of ruscoponin,
containing steroidal glycosides, were studied. In particular, it
causes lysis of fibrin in vitro (Kereselidze et al., 1975) and exhibits
a pronounced antiexudative effect, proving to be a low-toxic (LD50
tation pathways of different types of steroidal glycosides, and to
screen saponins belonging to different classes. Thus it can be used
to obtain rapid information about saponin composition of different
plants or parts of the same plant, allowing a rapid comparative
metabolite profiling of target matrixes.
3
.17 g/kg) phlebodynamic and antiexudative remedy (Mulkijanyan
Thereby, in order to fill the gap about R. ponticus composition,
we decided to carry out the phytochemical analysis of both the
underground parts and leaves of R. ponticus, with the double aim
to determine their main constituents and to ascertain the differ-
ences in their steroidal composition. In this way, 11 compounds
from R. ponticus underground parts, 3 of which new (5, 7–8), and
9 compounds from R. ponticus leaves, all found to be new com-
pounds (12–20), were identified.
and Abuladze, 1998, 2000). It is effective when administered either
systemically or locally, and does not reveal undesirable side effects
(Mulkijanyan and Abuladze, 1998, 2000).
Notwithstanding this, few phytochemical studies on R. ponticus
are reported in literature: so far only diosgenin and neoruscogenin
were found in the roots of R. ponticus (Pkheidze et al., 1971), along
with steroidal glycosides namely ruscoponticosides C, D, and E
(Korkashvili et al., 1985). Furthermore, there is only one report
about the steroidal composition of the leaves of R. ponticus
2
. Results and discussion
(Pkheidze et al., 1970).
To obtain a rapid comparative steroidal profiling of the under-
n
⇑
Corresponding author. Tel.: +39 089969763; fax: +39 089969602.
E-mail address: piacente@unisa.it (S. Piacente).
ground parts and leaves of R. ponticus, positive HPLC–ESIMS pro-
files of the ethanol extracts of both parts were obtained by using
0
031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2011.01.033

6
52
A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
+
+
the same analytical conditions (Fig. 1). Under these conditions, 11
chromatographic peaks (1–11) in the HPLC–ESIMS profile of
sequent losses of the sugar units from [(MꢀROH)+H] or [M+H]
n
+
and [M+Na] ions until to the aglycon ion peak (Table 1). In this re-
gard, it is noteworthy that most compounds displayed, in HPLC–
ESIMS as well as in HPLC–ESIMS , a diagnostic neutral loss of
144 or 142 a.m.u., from the [(M-162)+H] /[(M-162)+Na] ions
and from the [(MꢀROH-162)+H] ions (Table 1). This neutral loss
underground parts and 9 chromatographic peaks (12–20) in the
n
2
3
HPLC–ESIMS profile of leaves were displayed. A careful analysis
n
+
+
of ESIMS spectra recorded for each chromatographic peak allowed
+
us to preliminarily define the presence of at least two classes of
+
compounds, yielding the first abundant [(MꢀROH)+H] ions and
could be explained supposing the formation of the 7-hydroxy-6-
methylheptan-3-one or the 6-hydroxymethyl-hept-6-en-3-one
moiety, respectively, by the opening of the substituted E ring pres-
ent in spirostanol and dehydrated/or not furostanol glycosides.
This result led us to promptly ascertain the presence or not of an
exomethylene group on the aglycon moiety, and to easily distin-
guish spirostanol and dehydrated/or not furostanol glycosides
from the other steroidal glycosides. Thereby, it could be prelimi-
narily concluded that, while R. ponticus leaves was entirely charac-
terized by dehydrated/or not furostanol saponins, the underground
parts of the plant contained also spirostanol glycosides and two
compounds (8 and 9) belonging to classes of steroidal glycosides
differing from these latter. Moreover, the comparative analysis of
the full HPLC–ESIMS spectra of the chromatographic peaks due to
spirostanol and/or dehydrated furostanol glycosides interestingly
highlighted for one pair of compounds (7, 20) the same peculiarity
showed by furostanol glycosides, namely the same m/z value but
+
+
the second main [M+H] and [M+Na] ions (Table 1). This behavior
permitted us a first metabolite screening between furostanol
glycosides, possessing a labile hydroxy or methoxy group at
C-22 and thereby responsible for the formation of intense
+
[
(MꢀROH)+H] ions (1–4, 12–18) (Perrone et al., 2009), and steroi-
dal glycosides lacking in their structure of this labile group (5–11,
9–20). According to this analysis, the chromatographic profile of
1
each ethanol extract showed a clean metabolite separation, with
the furostanol glycosides eluting before the other steroidal glyco-
sides, and being much more present in the leaves extract than in
the underground parts. Analogously to what observed for
R. colchicus, when furostanol glycosides went along with their rel-
ative 22-methyl ethers, in both chromatograms pairs of peaks hav-
ing the same m/z value but different retention times were
displayed (12, 13 and 15, 16) (Table 1) (Perrone et al., 2009). The
n
analysis of the HPLC–ESIMS spectra of each chromatographic peak
n
allowed to add a piece in the complex puzzle of R. ponticus steroi-
dal composition. In fact, information about the number and the
nature (hexose/pentose) of sugar units as well as the aglycon moi-
ety could be obtained by the analysis of full and multistage HPLC–
different retention times (Table 1). The analysis of HPLC–ESIMS
spectra of this pair of peaks easily allowed to ascertain for them
a different glycosidation pattern and, thereby, a different aglycon
moiety. Finally, considering the results inferred from the HPLC–
n
n
ESIMS spectra of each chromatographic peak, observing the sub-
ESIMS data of underground parts and leaves of R. ponticus
Fig. 1. (a) HPLC–ESIMS profile of the ethanol extract of R. ponticus underground parts and (b) HPLC–ESIMS profile of the ethanol extract of R. ponticus leaves.

A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
653
Table 1
n
ESIMS and ESIMS product ions of compounds 5, 7, 8, 12–20 isolated from the underground parts and leaves of R. ponticus.
n
Compounds MW
MS1
1030 1053 [M+Na]+
MS fragment ions
+
+
+
+
+
5
7
891 [(M-162)+Na] , 749 [(M-162-142)+Na] , 745 [(M-162-146)+Na] , 729 [(M-162-162)+Na] , 613 [(M-162-146-132)+Na] ,
83 [(M-162-162-146)+Na] , 451 [(M-162-146-132-162)+Na]
747 [(M-146)+Na] , 731 [(M-162)+Na] , 615 [(M-146-132)+Na] , 587 [(M-162-144)+Na] , 453 [(M-146-132-162)+Na] , 441
(M-162-144-146)+Na] , 309 [(M-146-132-162-144)+Na]
+
+
5
893 [M+Na]+
+
+
+
+
+
870
+
+
[
661 [M+Na]+
901
617 [(M-44)+Na] , 515 [(M-146)+Na] , 383 [(M-146-132)+Na]
+
+
+
8
1
638
918
+
+
+
O)-162-144-146)+H]+,
2
2
3
4
5
6
7
8
9
0
739 [(MꢀH
2
O)-162)+H] , 595 [(MꢀH
2
O)-162-144)+H] , 593 [(MꢀH
2
O)-162-146)+H] , 449 [(MꢀH
+
+
+
[
9
(MꢀH
41 [M+Na]
901
(MꢀCH
2
O)+H]
431 [(MꢀH
2
O)-162-146-162)+H] , 287 [(MꢀH
2
O)-162-144-146-162)+H]
+
+
+
+
1
1
1
1
1
1
1
2
932
958
960
974
886
888
942
870
739 [(MꢀCH
3
OH)-162)+H] , 595 [(MꢀCH
3
OH)-162-144)+H] , 593 [(MꢀCH
3
OH)-162-146)+H] , 449 [(MꢀCH
3
OH)-162-144-
+
+
+
+
[
3
OH)+H]
146)+H] , 431 [(MꢀCH
3
OH)-162-146-162)+H] , 287 [(MꢀCH
3
OH)-162-144-146-162)+H]
+
9
55 [M+Na]
941
(MꢀH
+
+
+
+
795 [(MꢀH
2
O)-146)+H] , 779 [(MꢀH
2
O)-162)+H] , 735 [(MꢀH
2
O)-146-60)+H] , 637 [(MꢀH
2
O)-162-142)+H] , 633
O)-162-142-146)+H] , 431 [(MꢀH
+
+
+
+
[
9
2
O)+H]
81 [M+Na]
[(MꢀH
2
+
O)-162-146)+H] , 573 [(MꢀH
O)-146-60-162-162)+H]
2
O)-146-60-162)+H] , 491 [(MꢀH
2
2
O)-162-142-146-
+
+
60)+H] , 411 [(MꢀH
2
+
+
+
+
943
797 [(MꢀH
2
O)-146)+H] , 781 [(MꢀH
2
O)-162)+H] , 737 [(MꢀH
2
O)-146-60)+H] , 637 [(MꢀH
2
O)-162-144)+H] , 635
O)-162-144-146)+H] , 431 [(MꢀH
+
+
+
+
[(MꢀH
2
O)+H]
[(MꢀH
2
+
O)-162-146)+H] , 575 [(MꢀH
2
O)-162-146-60)+H] , 491 [(MꢀH
2
2
O)-162-144-146-
+
+
+
983 [M+Na]
60)+H] , 413 [(MꢀH
2
O)-162-146-60-162)+H] , 269 [(MꢀH
2
O)-162-144-146-60-162)+H]
+
+
+
+
943
(MꢀCH
797 [(MꢀCH
635 [(MꢀCH
3
OH)-146)+H] , 781 [(MꢀCH
3
OH)-162)+H] , 737 [(MꢀCH
3
OH)-146-60)+H] , 637 [(MꢀCH
OH)-162-146-60)+H] , 491 [(MꢀCH OH)-162-144-146)+H] , 431
OH)-162-146-60-162)+H] , 269 [(MꢀCH OH)-162-144-146-60-162)+H]
3
OH)-162-144)+H] ,
+
+
+
+
[
3
OH)+H]
3
OH)-162-146)+H] , 575 [(MꢀCH
3
3
+
+
+
+
9
97 [M+Na]
869
(MꢀH
[(MꢀCH
3
OH)-162-144-146-60)+H] , 413 [(MꢀCH
3
+
3
+
+
+
737 [(MꢀH
2
O)-132)+H] , 707 [(MꢀH
2
O)-162)+H] , 575 [(MꢀH
2
+
O)-162-132)+H] , 565 [(MꢀH
2
O)-162-142)+H] , 433
+
+
+
[
2
O)+H]
[(MꢀH
2
O)-162-142-132)+H] , 413 [(MꢀH
2
O)-162-132-162)+H] , 271 [(MꢀH
2
O)-162-142-132-162)+H]
+
909 [M+Na]
+
+
+
+
871
739 [(MꢀH
2
O)-132)+H] , 709 [(MꢀH
2
O)-162)+H] , 577 [(MꢀH
2
+
O)-162-132)+H] , 565 [(MꢀH
2
O)-162-144)+H] , 433
+
+
+
[
9
(MꢀH
2
O)+H]
[(MꢀH
2
O)-162-144-132)+H] , 415 [(MꢀH
2
O)-162-132-162)+H] , 271 [(MꢀH
2
O)-162-144-132-162)+H]
+
11 [M+Na]
+
+
+
+
+
+
943 [M+H]
797 [(M-146)+H] , 781 [(M-162)+H] , 737 [(M-146-60)+H] , 637 [(M-162-144)+H] , 635 [(M-162-146)+H] , 575 [(M-162-
46-60)+H] , 491 [(M-162-144-146)+H] , 431 [(M-162-144-146-60)+H] , 413 [(M-162-146-60-162)+H] , 269 [(M-162-144-
46-60-162)+H]
739 [(M-132)+H] , 709 [(M-162)+H] , 577 [(M-162-132)+H] , 565 [(M-162-144)+H] , 433 [(M-162-144-132)+H] , 415 [(M-
62-132-162)+H] , 271 [(M-162-144-132-162)+H]
+
+
+
+
1
+
1
871 [M+H]+
+
+
+
+
+
+
+
1
(
Table 1), and comparing them with those obtained from the
(1?2)-O-a-L-arabinopyranoside] (6) (Mimaki et al., 1996), the preg-
n
HPLC–ESIMS analysis of R. colchicus leaves (Perrone et al., 2009),
we could to claim that among all the 20 compounds detected in
both R. ponticus extracts only one, ruscoponticoside E (2), was al-
ready present in R. colchicus leaves.
At this point, to unambiguously elucidate these unknown
metabolites by NMR experiments, in particular ascertaining for
compounds 5–7, 10–11, and 19–20 the spirostanol or dehydrated
furostanol identity, all compounds from the underground parts
and leaves of R. ponticus were isolated and purified.
The analysis of positive HRMALDITOFMS spectrum of each com-
pound allowed to unambiguously assign them the respective
molecular formula. To determine the absolute configuration of
the sugar units, the crude saponin mixture has been submitted to
nane derivative namely 1b,3b-dihydroxypregna-5,16-dien-20-one
1-O- -rhamnopyranosyl-(1?2)-O- -arabinopyranoside (9)
(Bombardelli et al., 1972), along with two spirostanol derivatives
namely spirosta-5,25(27)-diene-1b,3b-diol 1-O-[b- -glucopyrano-
syl-(1?3)-O- -rhamnopyranosyl-(1?2)-O- -arabinopyrano-
side] (ruscoponticoside D) (10) (Korkashvili et al., 1985) and
spirosta-5,25(27)-diene-1b,3b-diol 1-O- -rhamnopyranosyl-
(1?2)-O- -arabinopyranoside (ruscoponticoside C) (11) (Kork-
ashvili et al., 1985).
a
-L
a-L
D
a
-
L
a-L
a-L
a-L
n
According to the HPLC–ESIMS results, the full positive ESIMS
spectrum of compound 5 was consistent with a not furostanolic
+
saponin, showing in fact as main peak the [M+Na] ion at m/z
n
1053 (Table 1). The analysis of the ESIMS spectra of 5 allowed
acid hydrolysis yielding
D
-glucose,
L
-rhamnose and
L
-arabinose;
to confirm this result and to ascertain the spirostanol or dehy-
drated furostanol nature of this compound, showing a characteris-
tic product ion at m/z 749, originating from the [(M-162)+Na]+
peak ion by neutral loss of the 6-hydroxymethyl-hept-6-en-3-
one moiety (142 a.m.u.). Moreover, the ESIMS spectrum of the
ion at m/z 1053 showed a fragmentation pattern in agreement with
the absolute configurations of the sugar units were established
by comparison of their optical rotation values with those reported
in the literature (Belitz et al., 2009; Wang et al., 2008).
By comparison of the spectroscopic data with literature values,
eight known compounds (1–4, 6, 9–11) were identified in R. ponticus
undergrounds, inparticularfivefurostanolderivativesnamely26-O-
2
the presence of one hexose, one deoxy-hexose, and one pentose
1
b-
O-[b-
-arabinopyranoside] (ruscoside) (1) (Bombardelli et al., 1972),
6-O-b- -glucopyranosyl-furosta-5,25(27)-diene-1b,3b,22 ,26-te-
trol 1-O- -rhamnopyranosyl-(1?2)-O- -arabinopyranoside
ruscoponticoside E) (2) (Bombardelli et al., 1972; Korkashvili
et al., 1985), 26-O-b- -glucopyranosyl-22 -methoxy-furosta-
,25(27)-diene-1b,3b,26-triol 1-O- -rhamnopyranosyl-(1?2)
O- -arabinopyranoside (3) (Mimaki et al., 1998), (25R)-26-O-b-
-glucopyranosyl-22 -methoxy-furost-5-ene-1b,3b,26-triol 1-O-
-rhamnopyranosyl-(1?2)-O- -arabinopyranoside (ceparoside
A) (4) (Yuan et al., 2008), 26-O-b- -glucopyranosyl-furosta-
,20(22),25(27)-triene-1b,3b,26-triol 1-O-[ -rhamnopyranosyl-
D
-glucopyranosyl-furosta-5,25(27)-diene-1b,3b,22
a
,26-tetrol 1-
moieties, as described in Table 1. The H NMR spectrum of 5
D-glucopyranosyl-(1?3)-O- -rhamnopyranosyl-(1? 2)-O-
a
-L
showed signals for three tertiary methyl groups at d 1.63 (3H, s),
1.13 (3H, s) and 0.75 (3H, s), exomethylene protons at d 5.93 and
4.96 (each 1H, br s), an olefinic proton at d 5.59 (1H, br d,
J = 5.7 Hz), three methine proton signals at d 4.74 (1H, dt, J = 10.1,
7.8, 5.3 Hz), 3.40 (1H, dd, J = 11.9, 3.9 Hz) and 3.38 (1H, m), indica-
tive of secondary alcoholic functions, two methylene proton sig-
nals at d 4.36 and 4.15 (each 1H, d, J = 12.1 Hz), ascribable to a
primary alcoholic function, along with four anomeric protons at d
5.33 (1H, d, J = 1.2 Hz), 4.57 (1H, d, J = 7.5 Hz), 4.31 (1H, d,
J = 7.5 Hz) and 4.30 (1H, d, J = 3.7 Hz) and a secondary methyl
a-L
2
D
a
a-
L
a-L
(
D
a
5
a-L
-
a-L
D
a
a-
L
a-L
1
3
D
group at d 1.29 (3H, d, J = 6.0 Hz). The C NMR spectrum displayed
2
5
a
-L
for the aglycon signals ascribable to six sp carbons at d 152.1,

6
54
A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
1
05.4, 146.4, 139.5, 125.7 and 112.7, three secondary alcoholic
osyl-(1?3)-O-
a-L-rhamnopyranosyl-(1?2)-O-a-L-
functions at d 85.7, 84.3 and 68.8, and one primary alcoholic func-
tion at d 72.1, suggesting the occurrence of a furostanol skeleton
arabinopyranoside].
The analysis of full and tandem mass experiments allowed to
assign compound 7 to the same saponin family of 5, showing as
only main differences the presence of the product ion originated
by the neutral loss of a 7-hydroxy-6-methylheptan-3-one moiety,
ascertaining the lack in 7 of an exomethylene group, a lower num-
ber of sugar units, and an aglycon moiety having a molecular
2
0,22
with a
D
double bond (Mimaki et al., 1996). The occurrence
double bond was confirmed from the HMBC spectrum
2
0,22
of a
D
which showed significative cross-peaks between the proton signal
of Me-21 (d 1.63) and C-20 (d 105.4)/C-22 (d 152.1). On the basis of
the HSQC and HMBC correlations, the aglycon moiety of compound
1
5
was identified as furosta-5,20(22),25(27)-triene-1b,3b,26-triol
weight 2 a.m.u. greater (Table 1). According to this result, the H
1
13
13
(Table 2). It was evident from the H and C NMR data that the su-
and C NMR data of aglycon portion of compound 7 in comparison
gar chain of 5 consisted of four sugar units. The chemical shifts of
all the individual protons of the four sugar units were ascertained
from a combination of 1D-TOCSY and DQF-COSY spectral analysis,
and the 1 C chemical shifts of their relative attached carbons were
assigned unambiguously from the HSQC spectrum (Table 5). These
data showed the presence of two b-glucopyranosyl units (d 4.57
to those of aglycon portion of 5 clearly suggested that 7 differed
from 5 only by the replacement of the exomethylene group with
H C
a secondary methyl group at C-27 (d 0.98, d 17.1). Thus, the
3
aglycon of 7 was established as (25R)-furosta-5,20(22)-diene-
1b,3b,26-triol. The C-25 configuration was deduced to be R based
on the difference of chemical shifts (
D
ab = d
a
ꢀ d
b
) of the geminal
and 4.31), one
a-arabinopyranosyl (d 4.30) and one
a-rhamnopyr-
protons at H -26 ( ab = 0.34 ppm). It has been described that dab
2
D
anosyl unit (d 5.33). The
a
configuration of the rhamnopyranosyl
is usually >0.57 ppm in 25S compounds and <0.48 in 25R com-
pounds (Agrawal, 2004). The 25R configuration was confirmed by
hydrolysis of compound 7 with b-glucosidase providing the corre-
sponding spirostanol glycoside. The NMR data of its aglycon moi-
ety were in agreement with those reported for (25R)-spirost-5-
ene-1b,3b-diol or ruscogenin (Agrawal et al., 1985).
unit was deduced from the value of JH1–H2 coupling (J = 1.2 Hz)
and from the absence of intraresidual ROESY correlations between
H-1rha and H-3rha/H-5rha. It was also confirmed by the H-1/C-1 J
value = 169 Hz, measured from the residual direct correlation
observed in the HMBC spectrum, in agreement with that reported
for the alpha anomer of rhamnopyranose (Kasai et al., 1979). Gly-
cosidation shifts were observed for C-1 (d 84.3), C-26 (d 72.1), C-
A detailed comparison of NMR data of sugar portion of com-
pound 7 with those of compound 5 showed the absence of the
3
rha (d 82.6) and C-2ara (d 75.3). An unambiguous determination
b-glucopyranosyl unit at C-3 of
fore, compound 7 was identified as the new (25R)-26-O-b-
pyranosyl-furosta-5,20(22)-diene-1b,3b,26-triol 1-O-[
rhamnopyranosyl-(1?2)-O- -arabinopyranoside].
a
-rhamnopyranosyl unit. There-
of the sequence and linkage sites was obtained from the HMBC
spectrum, which showed key correlation peaks between the proton
signal at d 4.30 (H-1ara) and the carbon resonance at d 84.3 (C-1), d
D-gluco-
a-L-
a-L
n
5
.33 (H-1rha) and d 75.3 (C-4ara), d 4.57 (H-1glcI) and d 82.6 (C-3rha),
In agreement with HPLC–ESIMS results, the analysis of ESIMS
n
and the proton signal at d 4.31 (H-1glcII) and the carbon resonance
at d 72.1 (C-26). On the basis of all these evidences, the structure of
the new compound 5 was established as 26-O-b-
and ESIMS spectra of 8 provided to assign it to a saponin class dif-
+
ferent from the furostanolic, lacking the typical [(MꢀROH)+H] ion
D-glucopyranosyl-
peak, and from compounds 5 and 7, which showed product ions
furosta-5,20(22),25(27)-triene-1b,3b,26-triol 1-O-[b-
D
-glucopyran-
originated by neutral loss of 142 or 144 a.m.u. On the contrary,
Table 2
1
3C and 1H NMR data (J in Hz) of the aglycon moieties of compounds 5, 7 and 8 (600 MHz, CD
3
OD).
5
7a
8
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
84.3
37.4
68.8
43.4
3.40 dd (11.9, 3.9)
2.16, 1.69 m
3.38 m
2.28, 2.24 m
–
5.59 br d (5.7)
1.99, 1.56 m
1.56 m
1.28 m
–
2.58, 1.47 m
1.75, 1.34 m
–
84.1
37.3
69.0
43.2
139.4
125.7
32.6
33.8
51.2
43.3
24.6
41.0
44.1
56.2
34.9
85.2
65.5
14.6
15.1
105.5
11.5
152.7
24.0
31.3
34.1
75.7
3.53 dd (11.9, 3.9)
2.11, 1.71 m
3.39 m
2.27, 2.24 m
–
5.59 br d (5.7)
1.99, 1.57 m
1.55 m
1.38 m
–
2.53, 1.47 m
1.73, 1.34 m
–
84.3
37.1
68.8
43.2
3.40 dd (11.9, 3.9)
2.13, 1.71 m
3.38 m
2.28, 2.24 m
–
5.59 br d (5.7)
2.01, 1.58 m
1.57 m
1.32 m
–
2.64, 1.49 m
1.76, 1.26 m
–
1.18 m
2.32, 1.50 m
5.04 m
1.99 d (7.5)
0.80 s
1.14 s
2.65 q (7.5)
1.32 d (7.5)
–
139.5
125.7
32.6
34.2
51.2
43.2
24.5
40.7
43.7
56.2
35.6
85.7
65.9
14.5
15.1
105.4
11.4
152.1
24.6
31.0
146.4
72.1
139.4
125.3
32.4
33.8
51.2
43.2
24.0
39.1
41.9
55.7
34.0
84.5
60.1
14.3
14.9
37.5
17.9
184.1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1.08 m
1.12 m
2.20, 1.44 m
4.74 dt (10.1, 7.8, 5.3)
2.51 d (10.1)
0.75 s
1.13 s
–
1.63 s
–
2.30 (2H) m
2.29 (2H) m
–
2.18, 1.44 m
4.74 dt (10.1, 7.8, 5.3)
2.51 d (10.1)
0.75 s
1.12 s
–
1.64 s
–
2.16, 1.37 m
1.65, 1.41 m
1.79 m
4.36 d (12.1)
3.76, 3.42 m
4
.15 d (12.1)
5.13 br s
.96 br s
2
7
112.7
17.1
0.98 d (6.6)
4
a
The chemical shift values of the aglycon moiety of 19 deviate from the experimental values of 7 of ±0.03 ppm.

A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
655
2
the ESIMS spectrum highlighted the presence of a product ion at
m/z 617, due to the neutral loss of 44 a.m.u. due to a CO moiety,
This evidence was confirmed by the HMBC correlations between
the proton signals at d 4.35 and 4.31 (H -6glcI) and d 2.08 and the
carbon resonance at d 172.3 (COCH ). Once again, the sugar se-
2
2
together with two product ions, at m/z 515 and 383, due to consec-
3
utive neutral losses of one deoxy-hexose and one pentose sugar.
quence and the linkage sites were deduced from HSQC and HMBC
experiments. The glycosidation shifts on C-1 (d 84.1), C-26 (d 75.7)
and C-2glcI (d 79.6) indicated the linkage sites. In the HMBC spec-
trum key correlation peaks between the proton signal at d 4.38
ꢀ1
The IR spectrum of 8 showed an absorption peak at 1745 cm
1
due to a carbonyl group. For the aglycon portion in the H NMR
spectrum (Table 2) two tertiary methyl groups at d 1.14 (3H, s)
and 0.80 (3H, s), a secondary methyl group at d 1.32 (3H, d,
J = 7.5 Hz), an olefinic proton at d 5.59 (1H, br d, J = 5.5 Hz), three
methine proton signals at d 5.04 (1H, m), 3.40 (1H, dd, J = 11.9,
(H-1glcI) and the carbon resonance at d 84.1 (C-1), d 5.37 (H-1rha
)
and d 79.6 (C-2glcI), and the proton signal at d 4.28 (H-1glcII) and
the carbon resonance at d 75.7 (C-26) were observed. Therefore,
3
.9 Hz) and 3.38 (1H, m), indicative of secondary alcoholic func-
compound 19 was established as the new (25R)-26-O-b-
pyranosyl-furosta-5,20(22)-diene-1b,3b,26-triol 1-O-[ -rhamno-
pyranosyl-(1?2)-6-O-acetyl-b- -glucopyranoside].
Full positive ESIMS profile of 15 was in agreement with a furost-
D-gluco-
tions, were observed. The NMR data of the aglycon portion of 8
in comparison to those reported for vespertilin (Gonzalez et al.,
1
by the presence of a secondary alcoholic function to C-1. HBMC
correlations between H-16 (d 5.04), H-17 (d 1.99), H-20 (d 2.65),
Me-21 (d 1.32) and the carbonyl group at d 184.1 along with the
downfield shifts of H-16 (d 5.04) and C-16 (d 84.5) signals con-
firmed the presence of a five-membered lactone ring between C-
a-L
D
971) revealed that the aglycon of 8 differed from vespertilin only
+
anol structure, showing the diagnostic [(MꢀH
2
O)+H] ion as main
peak. Analogously to 19, the analysis of ESIMS spectra of 15 allowed
n
to ascertain once again the presence of an acetyl moiety (Table 1).
1
The H NMR spectrum of 15 showed signals for two tertiary methyl
groups at d 1.12 (3H, s) and 0.87 (3H, s), three secondary methyl
groups at d 1.28 (3H, d, J = 6.0 Hz), 1.04 (3H, d, J = 6.6 Hz) and 0.98
(3H, d, J = 6.6 Hz), an olefinic proton at d 5.59 (1H, br d, J = 5.7 Hz),
three methine proton signals at d 4.40 (1H, m), 3.53 (1H, dd,
J = 11.9, 3.9 Hz) and 3.39 (1H, m), indicative of secondary alcoholic
functions, and two methylene proton signals at d 3.77 and 3.42 (each
1H, m), ascribable to a primary alcoholic function, along with three
anomeric protons at d 5.37 (1H, d, J = 1.2 Hz), 4.38 (1H, d, J = 7.5 Hz)
and 4.28 (1H, d, J = 7.5 Hz). The comparison of NMR data of com-
pound 15 with those of compound 19 revealed that compound 15
was the 22-hydroxy derivative of compound 19. Thus, on the basis
of the HSQC and HMBC correlations, the aglycon moiety of com-
2
2 and C-16. Additionally, the relative configuration of C-20 was
derived by the NOE correlations between H-14 (d 1.18), H-16 (d
.04) and H-17 (d 1.99) signals, between H-16 (d 5.04) and H-17
d 1.99) signals, between H-17 (d 1.99) and Me-21 (d 1.32) signals
a
5
(
and between Me-18 (d 0.80) and H-20 (d 2.65) signals. On the basis
of these data the aglycon of 8 was identified as the new vespertilin
derivative (20S)-1b,3b,16b-trihydroxypregn-5-ene-20-carboxylic
acid 22,16-lactone. Vespertilin, also reported as diosgeninlactone,
has been isolated for the first time from the ethanolic extract of
the fruits of Solanum vespertilio (Gonzalez et al., 1971). Products
containing the 22,16-c-lactone moiety have been isolated from dif-
ferent vegetable sources and postulated to be metabolic products
of the corresponding sapogenins. Thus, lactone-type sapogenols
and their glycosides might be biosynthetically derived from the
genuine 23,26-oxygenated spirostane- or furostane-type (Nafady
pound 15 was identified as furosta-5,25(27)-diene-1b,3b,22
trol. The configuration of the hydroxy group
C-22 was established to be from ROESY correlations between H-
20 (d 2.22) and the protons H-23a (d 1.85) and H-23b (d 1.65).
Therefore, the structure of compound 15 was identify as (25R)-26-
a
,26-te-
at
a
1
et al., 2003). Additionally, the H NMR of compound 8 displayed
signals for two anomeric proton at d 5.33 (1H, d, J = 1.2 Hz) and
O-b-
D
-glucopyranosyl-furost-5-ene-1b,3b,22
-glucopyran oside].
As well as for compound 15, ESIMS data of 12 provided infor-
mation about its furostanol nature, showing a characteristic
a
,26-tetrol
1-O-[a-
4
1
.30 (1H, d, J = 3.7 Hz) along with a secondary methyl group at d
.28 (3H, d, J = 6.0 Hz). Comparison of NMR data of the sugar por-
L-rhamnopyranosyl-(1?2)-6-O-acetyl-b-D
n
tion of compound 8 with those of compound 7 showed that the
disaccharide chain at C-1 of the aglycon portion was identical in
the two compounds. Thus, compound 8 was identified as the
new (20S)-1b,3b,16b-trihydroxypregn-5-ene-20-carboxylic acid
+
[(MꢀH
2
O)+H] ion at m/z 901, and the type of sugar units, yielding
product ions due to consecutive neutral losses of two hexose and
one deoxy-hexose sugar. The NMR data of 12 were superimposable
with those of compound 15 except for the absence of the acetyl
group at C-6 of the glucopyranosyl unit linked at C-1 of the aglycon
2
2,16-lactone 1-O-[
a-
L-rhamnopyranosyl-(1?4)-O-b-D-glucopy-
ranoside].
n
The analysis of ESIMS and ESIMS spectra of 19 showed in the
(Table 5). Thus, compound 12 was identified as (25R)-26-O-b-
glucopyranosyl-furost-5-ene-1b,3b,22 ,26-tetrol 1-O-[ -rha-
mnopyranosyl-(1?2)-O-b- -glucopyranoside].
The full ESIMS spectra of 16 and 13 clearly identified these com-
D-
+
full ESIMS spectrum the [M+H] ion peak at m/z 943, and in the
a
a-L
n
+
ESIMS spectra the [(M-162–144)+H] product ion at m/z 637 along
D
with the product ions originated from the latter by consecutive
n
+
neutral loss of sugar unit (Table 1). Interestingly, the ESIMS frag-
pounds as furostanol-type, showing the [(MꢀCH
3
OH)+H] ion as
mentation pattern allowed us to identify in 19 the presence of an
acetyl moiety, yielding product ions formed by neutral loss of
the main peak for both. They could be defined as the 22-methyl
ether derivatives of 15 and 12, respectively, each pair showing
the same value of m/z, but differing for the m/z of the relative
6
0 a.m.u (Table 1). According to mass spectrometric results, the
1
13
+
H and C NMR chemical shifts of the aglycon moieties of 19
and 7 were almost superimposable (see Table 2) confirming the
same aglycon portion. Moreover, for 19 the H NMR spectrum dis-
[M+Na] ion, greater of 14 a.m.u. (Table 1). In particular, it could
be asserted that 16 and 13 were naturally occurring compounds
in R. ponticus leaves ruling out they were artefacts derived from
compounds 12 and 15 being detectable in the HPLC–ESIMS profile
of the ethanol extract performed without methanol in the mobile
phase. This result was confirmed by NMR data ( H, C, 1D-TOCSY,
DQF-COSY, HSQC, HMBC, ROESY) of compounds 16 and 13, being
apparent that these compounds differed from 15 and 12, respec-
tively, only by the presence of a methoxy group instead of a hydro-
xy group at C-22 (Table 3). Therefore, compound 16 was deduced
1
played signals for three anomeric protons at d 5.37 (1H, d,
J = 1.2 Hz), 4.38 (1H, d, J = 7.5 Hz) and 4.28 (1H, d, J = 7.5 Hz) along
with a secondary methyl group at d 1.28 (3H, d, J = 6.0 Hz) and a
singlet signal at d 2.08 (3H, s) ascribable to the methyl group of
1
13
1
13
an acetyl group. Complete assignments of the H and C NMR sig-
nals of the sugar portion were accomplished by HSQC, HMBC, DQF-
COSY and 1D-TOCSY experiments which led to the identification of
one
d 4.28) and one 6-O-acetyl-b-glucopyranosyl (d 4.38) unit. The
presence of the acetyl group was suggested by the downfield shifts
observed for H -6glcI (d 4.35 and 4.31) and C-6glcI (d 65.0) (Table 5).
a
-rhamnopyranosyl (d 5.37) unit, one b-glucopyranosyl
to be (25R)-26-O-b-
ene-1b,3b,26-triol 1-O-[
b- -glucopyranoside and compound 13 was established as
(25R)-26-O-b- -glucopyranosyl-22 -methoxy-furost-5-ene-1b,
D
-glucopyranosyl-22
a-methoxy-furost-5-
(
a-L-rhamnopyranosyl-(1?2)-6-O-acetyl-
D
2
D
a

6
56
A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
Table 3
1
3C and 1H NMR data (J in Hz) of the aglycon moieties of compounds 14, 15 and 16 (600 MHz, CD
3
OD).
1
4
15a
16a
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
84.2
36.9
69.0
43.2
3.53 dd (11.9, 3.9)
2.10, 1.72 m
3.39 m
2.28, 2.24 m
–
5.58 br d (5.7)
1.99, 1.58 m
1.57 m
1.38 m
–
2.58, 1.48 m
1.72, 1.25 m
–
84.2
37.2
68.9
43.2
139.3
125.8
32.5
33.9
51.0
43.0
24.9
41.0
40.5
57.6
32.6
82.4
64.9
16.6
14.7
40.9
15.7
113.9
31.3
28.9
34.7
75.9
3.53 dd (11.9, 3.9)
2.11, 1.71 m
3.39 m
2.27, 2.24 m
–
5.59 br d (5.7)
2.00, 1.55 m
1.57 m
1.38 m
–
2.51, 1.46 m
1.69, 1.25 m
–
84.1
37.1
68.8
43.2
139.4
125.7
32.4
34.0
51.0
43.4
24.4
40.9
40.6
57.6
32.6
82.1
65.0
16.9
15.1
40.9
16.0
113.7
31.3
29.0
34.8
75.7
3.53 dd (11.9, 3.9)
2.10, 1.72 m
3.39 m
2.27, 2.24 m
–
5.58 br d (5.7)
1.99, 1.57 m
1.57 m
1.38 m
–
2.52, 1.47 m
1.69, 1.24 m
–
139.5
125.7
32.4
33.8
51.2
43.1
24.6
41.0
40.9
57.6
32.5
82.3
65.1
16.8
14.9
41.0
15.8
113.4
32.1
28.2
146.9
72.4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1.20 m
2.00, 1.32 m
4.40 m
1.76 m
0.88 s
1.21 m
2.00, 1.32 m
4.40 m
1.77 m
0.87 s
1.20 m
1.99, 1.31 m
4.39 m
1.76 m
0.87 s
1.12 s
2.21 m
1.12 s
2.22 m
1.12 s
2.22 m
1.04 d (6.6)
–
1.93, 1.86 m
2.32, 2.23 m
–
1.04 d (6.6)
–
1.85, 1.65 m
1.61, 1.17 m
1.77 m
1.04 d (6.6)
–
1.86, 1.65 m
1.64, 1.19 m
1.77 m
4.37 d (12.1)
3.77, 3.42 m
3.76, 3.41 m
4
.16 d (12.1)
5.12 br s
.96 br s
2
7
111.6
16.9
–
0.98 d (6.6)
17.3
47.4
0.98 d (6.6)
3.17 s
4
OCH
3
a
The chemical shift values of the aglycon moieties of 12 and 13 deviate from the experimental values of 15 and 16 of ±0.03 ppm,
respectively.
3
b,26-triol 1-O-[
a
-
L
-rhamnopyranosyl-(1?2)-O-b-
D
-glucopyrano-
in good agreement with those reported for (25R)-furosta-5,20(22)-
diene-3b,26-diol or pseudodiosgenin (Liu and Chen, 2002). The H
1
side. Compounds 16 and 13 are 22-O-methyl ethers derivatives
of compounds 15 and 12, respectively.
NMR of the sugar region displayed signals for three anomeric pro-
tons at d 4.45 (1H, d, J = 7.5 Hz), 4.33 (1H, d, J = 3.7 Hz) and 4.27
(1H, d, J = 7.5 Hz). On the basis of 1D-TOCSY, HSQC, HMBC,
The analysis of positive full and multistage mass spectra of 14,
ascertaining its furostanol nature, sugar composition, and acety-
lated form, allowed to identify the aglycon moiety, resulting of
DQF-COSY correlations these sugar units was identified as a-arabi-
2
a.m.u. smaller than that of 15 (Table 1). This result was indicative
nopyranose (d 4.33) and b-glucopyranose (d 4.45 and 4.27). The
linkage sites were determined by the HMBC spectrum, which
showed key correlation peaks between the proton signal at d
4.45 (H-1glcI) and the carbon resonance at d 79.7 (C-3), d 4.33 (H-
of the presence of an exomethylene group, as supported by the
occurrence of product ions obtained by neutral loss of the 6-
hydroxymethyl-hept-6-en-3-one moiety (142 a.m.u.). Accordingly,
a detailed analysis of NMR data of compound 14 in comparison
with those of compound 15 confirmed that the two compounds
differed only by the presence of a secondary methyl group at C-
1
ara) and d 80.4 (C-4glcI) and the proton signal at d 4.27 (H-1glcII
and the carbon resonance at d 75.7 (C-26). On the basis of this evi-
dence the new compound 20 was deduced to be (25R)-26-O-b-
glucopyranosyl-furosta-5,20(22)-diene-3b,26-diol 3-O-[ -arabi-
nopyranosyl-(1? 4)-O-b- -glucopyranoside].
)
D
-
2
7 (d
H
0.98, d
C
17.1) instead of the exomethylene group (Table 3).
a-L
Following the same method of analysis shown for compound 7 to
determine the C-25 configuration, the structure of compound 14
D
The analysis of full ESIMS spectrum provided to identify
was established as (25R)-26-O-b-
b,3b,22 ,26-tetrol 1-O-[ -rhamnopyranosyl-(1?2)-6-O-acetyl-
b- -glucopyranoside].
The full ESIMS spectrum of compound 20 showed as main peak
D
-glucopyranosyl-furost-5-ene-
compound 18 as a furostanol glycoside, showing the typical
+
1
a
a-
L
[(MꢀH
2
O)+H] ion at m/z 871. Further information about the struc-
ture and the sugar units could be obtained by the analysis of
D
n
ESIMS data, highlighting the lacking of exomethylene function
+
the [M+H] ion at m/z 871, suggesting a no furostanolic structure.
and the presence of two hexose and one pentose sugar (Table 1).
n
1
13
Besides, the finding in ESIMS spectra of the product ion originated
In agreement with these results, the H and C NMR data of com-
pound 18 in comparison with those of compound 20 showed that
compound 18 was the 22-hydroxy derivative of compound
20 (Table 4). Thus, compound 18 was identified as (25R)-26-O-b-
+
by neutral loss of 144 a.m.u. from the [(M-162)+H] ion, allowed to
1
claim the presence of a methyl group at C-27. The H NMR spec-
trum of 20 showed for the aglycon portion signals for three tertiary
methyl groups at d 1.64 (3H, s), 1.08 (3H, s) and 0.75 (3H, s), one
secondary methyl group at d 0.97 (1H, d, J = 6.6 Hz), an olefinic pro-
ton at d 5.41 (1H, br d, J = 5.7 Hz), two methine proton signals at
D-glucopyranosyl-furost-5-ene-3b,22a,26-triol 3-O-[a-L-arabino-
pyranosyl-(1?4)-O-b- -glucopyranoside].
D
Finally, the analysis of full and multistage mass spectra ac-
4
.74 (1H, dt, J = 10.1, 7.8, 5.3 Hz) and d 3.60 (1H, m) indicative of
quired for compound 17 allowed to ascertain only one structural
25,27
secondary alcoholic functions, and two methylene proton signals
at d 3.75 and 3.42 (each 1H, m), ascribable to a primary alcoholic
function. The NMR data of the aglycon moiety of 20 (Table 4) were
difference between this latter and 18, being the first the
D
-
dehydro-derivative of the second. In fact, compound 17 displayed
+
the [(MꢀH
2
O)+H] ion and the main product ions of 2 a.m.u

A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
657
1
n
smaller than the corresponding ions in 18. The NMR data of com-
pound 17 confirmed that this compound differed from 18 only
by the replacement of the secondary methyl group at C-27 with
an exomethylene group (Table 4). Therefore, the structure of
acquired in MS and MS scanning modes. By using a syringe pump
2 3
(flow rate 5 ll/min), each pure compound dissolved in H O/CH CN
(1:1) was infused in the ESI source. Positive ESIMS analyses were
performed using the same conditions as those for HPLC–ESIMS
n
n
compound 17 was deduced to be 26-O-b-
sta-5,25(27)-diene-3b,22 ,26-triol 3-O-[
1?4)-O-b- -glucopyranoside].
In conclusion, the so far described analytical methodology al-
D
-glucopyranosyl-furo-
analysis.
a
a
-L
-arabinopyranosyl-
Semi-preparative HPLC separations were carried out on a
Waters 590 system equipped with a Waters R401 refractive index
detector, a Waters XTerra Prep MSC18 column (300 ꢁ 7.8 mm i.d.)
and a Rheodyne injector.
(
D
lowed us to clarify the saponin composition of the underground
parts and leaves of R. ponticus. In particular, the adopted HPLC–
n
ESIMS method resulted to be able to give a rapid comparative
3.2. Plant material
metabolite profiling of these different parts of the plant, promptly
highlighting their similarity and their differences. The careful
study of HPLC–ESIMS data of each chromatographic compound al-
The underground parts and leaves of R. ponticus Wor. were col-
lected in June of 2007 in the area of Tbilisi, in Georgia. Samples of R.
ponticus were identified by Dr. Jemal Aneli, Department of Botany,
Institute of Pharmacochemistry, Tbilisi, Georgia. A voucher speci-
men (No. 368) has been deposited at this Department.
n
lowed us to obtain an easy and quick screening of the different
type of saponins occurring in this Ruscus species, underlining a
clear prevalence of furostanol glycoside derivatives in R. ponticus
leaves rather in the underground parts of the plant, showing this
latter a wider structure variety. Moreover, from these results, a
characteristic chromatographic elution trend for R. ponticus sapo-
nins could be drawn, first eluting the furostanol glycosides, fol-
lowed by their dehydrated forms and by vespertilin derivatives,
and finally by pregnane and spirostanol glycosides. In particular,
the furostanol glycosides isolated from R. ponticus are mainly furo-
3.3. Extraction and isolation
The air-dried, powdered underground parts of R. ponticus
(100 g) were extracted with 70% EtOH, three times at 60 °C. The
collected alcohol-aqueous extract was concentrated (21 g) and
then suspended in water and partitioned with n-BuOH. The BuOH
extract was dried under vacuum (8 g). Part of extract (3 g) was sub-
jected to Sephadex LH-20 chromatography, eluting with MeOH to
yield 11 combined fractions.
sta-5,25(27)-diene-1b,3b,22
a
,26-tetrol,
furosta-5,25(27)-diene-
3
b,22 ,26-triol and furosta-5,20(22),25(27)-triene-1b,3b,26-triol
a
derivatives along with the corresponding (25R)-25,27-dihydro-
derivatives. Interestingly, the occurrence of dehydrated furostanol
derivatives, for the first time isolated from a Ruscus species, is an
unusual finding which makes unique the saponins profile of R.
ponticus.
Fractions 29–38 and 51–61 were chromatographed by RP-HPLC
(Waters XTerra Prep MSC18 column, 300 ꢁ 7.8 mm i.d.), at flow
2
rate 2.0 ml/min, using different mixtures of MeOH:H O in isocratic
conditions.
Fraction 29–38 (297.8 mg) was chromatographed by RP-HPLC
using MeOH–H O (50:50) as mobile phase to yield compound 5
(1.2 mg, t = 32.8 min). From fraction 51–61 (325.1 mg) com-
pounds 1 (1.3 mg, t = 28.4 min), 2 (1.2 mg, t = 32.0 min), 3
(1.0 mg, = 33.0 min), (1.1 mg, = 33.5 min), (7.8 mg,
= 45.8 min), and 7 (3.4 mg, t = 46.5 min) were obtained by RP-
HPLC using MeOH–H O (40:60) as mobile phase.
2
3
. Experimental
R
R
R
3.1. General
t
R
4
t
R
6
t
R
R
Optical rotations were measured on a JASCO DIP 1000 polarim-
2
eter. IR measurements were obtained on a Bruker IFS-48 spectrom-
eter. NMR experiments were performed on a Bruker DRX-600
spectrometer (Bruker BioSpinGmBH, Rheinstetten, Germany)
equipped with a Bruker 5 mm TCI CryoProbe at 300 K. All 2D-
The remaining 5 g of BuOH extract of the underground parts
was subjected to silica gel column chromatography (120 ꢁ 3 cm,
100/160 lm, Merck), eluting with isocratic system chloro-
form:methanol:water (26:14:3), to yield three combined fractions
with different polarity: apolar fraction (0.9 g), media polar (2.6 g)
and polar fraction (1.3 g).
3
NMR spectra were acquired in CD OD (99.95%, Sigma–Aldrich)
and standard pulse sequences and phase cycling were used for
DQF-COSY, HSQC, HMBC and ROESY spectra. The NMR data were
processed using UXNMR software. Exact masses were measured
by a Voyager DE mass spectrometer equipped with a 337 nm laser
and delay extraction and operated in positive ion reflector mode.
Samples were analyzed by MALDITOF mass spectrometry. A mix-
Apolar fraction was chromatographed by RP-HPLC (flow rate
2.0 ml/min) using MeOH–H
compounds 8 (1.4 mg, t = 32.5 min), and 9 (2.6 mg, t
and using MeOH–H O (66:34) as mobile phase to yield compounds
10 (1.2 mg, t = 6.2 min) and 11 (1.2 mg, t = 7.4 min).
2
O (54:46) as mobile phase to yield
R
R
= 40.2 min),
2
R
R
ture of analyte solution and
a
-cyano-4-hydroxycinnamic acid (Sig-
The air-dried, powdered leaves of R. ponticus (100 g) were ex-
tracted with 70% EtOH, three times at 60 °C. The collected alco-
hol-aqueous extract was concentrated (16 g) and suspended in
water and successively partitioned with chloroform/n-BuOH. The
BuOH extract was dried under vacuum (10 g).
ma) was applied to the metallic sample plate and dried. Mass
calibration was performed with the ions from adrenocorticotropic
hormone (ACTH) fragment 18–39 human at 2465.1989 Da and
a-cyano-4-hydroxycinnamic acid at 190.0504 Da as internal stan-
n
dard. Ethanol extract was analyzed by on-line HPLC–ESIMS using
a ThermoFinnigan Spectra System HPLC coupled with an LCQ Deca
ion trap mass spectrometer (ThermoFinnigan, San Jose’, CA, USA).
HPLC separation was conducted on a C18 reversed-phase (RP) col-
Part of the extract (3 g) was subjected to Sephadex LH-20 chro-
matography, eluting with MeOH to yield 16 combined fractions.
Fractions 17, 18, and 19–21 were chromatographed by RP-HPLC
(Waters XTerra Prep MSC18 column, 300 ꢁ 7.8 mm i.d.), at flow
umn (5
MA) at a flow rate of 0.2 ml/min. A gradient elution was performed
by using H O (A) and CH CN (B) as mobile phases, from 20% B to
0% B in 25 min. The column effluent was analyzed by ESIMS in po-
lm, 2.1 mm ꢁ 250 mm; X-Terra MS C18; Waters, Milford,
2
rate 2.0 ml/min, using different mixtures of MeOH:H O in isocratic
conditions.
2
3
Fraction 17 (220.5 mg) was chromatographed by RP-HPLC using
MeOH–H O (40:60) as mobile phase to yield compounds 13
(1.9 mg, t = 26.8 min) and 16 (1.6 mg, t = 30.0 min). Fraction 18
(239.7 mg) was chromatographed by RP-HPLC using MeOH–H
(40:60) as mobile phase to yield compounds 12 (1.4 mg,
= 26.0 min), 14 (1.2 mg, t = 29.0 min), 15 (1.6 mg, t = 29.6 min),
7
2
sitive ion mode and the mass spectra were acquired and processed
using the software provided by the manufacturer. The capillary
voltage was set at 23 V, the spray voltage at 5 kV and the tube lens
offset at 50 V. The capillary temperature was 280 °C. Data were
R
R
2
O
t
R
R
R

6
58
A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
Table 4
1
3C and 1H NMR data (J in Hz) of the aglycon moieties of compounds 17, 18 and 20 (600 MHz, CD
3
OD).
17
18
20
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
38.4
30.4
79.6
39.4
1.92, 1.12 m
1.96, 1.34 m
3.60 m
2.47, 2.30 m
–
5.42 br d (5.7)
2.00, 1.60 m
1.71 m
1.02 m
–
1.59 (2H) m
1.82, 1.23 m
–
1.18 m
2.00, 1.32 m
4.40 m
1.77 m
0.87 s
1.09 s
2.22 m
1.04 d (6.6)
38.4
30.4
79.6
39.6
141.9
122.4
32.6
32.7
51.3
39.0
21.8
40.5
41.7
57.6
32.7
82.3
64.9
16.8
19.8
41.0
16.0
114.3
31.3
28.5
34.7
75.7
17.1
1.91, 1.11 m
1.96, 1.37 m
3.60 m
2.46, 2.29 m
–
5.41 br d (5.7)
1.99, 1.57 m
1.70 m
1.01 m
–
1.59 (2H) m
1.83, 1.24 m
–
38.3
30.5
79.7
39.6
1.91, 1.11 m
1.96, 1.33 m
3.60 m
2.46, 2.29 m
–
5.41 br d (5.7)
2.06, 1.59 m
1.70 m
1.00 m
–
1.59 (2H) m
1.82, 1.20 m
–
141.7
122.3
32.6
32.6
51.6
38.0
21.8
40.5
41.5
57.4
32.6
82.1
64.9
16.8
19.6
41.0
16.2
113.4
32.1
28.2
147.3
72.4
112.0
142.1
122.5
32.9
32.5
51.6
38.0
21.8
40.7
43.4
56.2
34.9
85.2
65.5
14.6
19.6
105.3
11.6
152.7
23.8
31.1
34.1
75.7
17.1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
1.18 m
2.02, 1.32 m
4.40 m
1.76 m
0.87 s
1.12 m
2.18, 1.45 m
4.74 dt (10.1, 7.8, 5.3)
2.52 d (10.1)
0.75 s
1.08 s
–
1.08 s
2.21 m
1.04 d (6.6)
–
1.85, 1.65 m
1.62, 1.19 m
1.77 m
1.64 s
–
1.93, 1.86 m
2.32, 2.23 m
–
4.37 d (12.1) 4.15 d (12.1)
5.12 br s 4.96 br s
2.15, 1.37 m
1.66, 1.41 m
1.79 m
3.75, 3.42 m
0.97 d (6.6)
3.76, 3.42 m
0.98 d (6.6)
1
7 (1.3 mg, t
fraction 19–21 (384.1 mg) compounds 19 (1.1 mg, t
and 20 (1.4 mg, t = 35.0 min) were obtained by RP-HPLC using
MeOH–H O (54:46) as mobile phase.
R
= 40.2 min), and 18 (1.1 mg, t
R
= 40.8 min). From
3.7. (25R)-26-O-b-
D
-glucopyranosyl-furost-5-ene-1b,3b,22
a,26-tetrol
R
= 30.6 min)
1-O-[ -rhamnopyranosyl-(1?2)-O-b-D
a
-L
-glucopyranoside] (12)
R
2
D
2
2
Amorphous white solid; C45
H
74
O19; ½
a
ꢂ
ꢀ40.8° (c 0.1 MeOH);
KBr
ꢀ1
13
IR
m
cm : 3479 (>OH), 2950 (>CH), 1270 and 1051 (C–O–C);
max
1
for H and C NMR (CD
3
OD, 600 MHz) data of the aglycon moiety
3.4. 26-O-b-
D
-glucopyranosyl-furosta-5,20(22),25(27)-triene-
-glucopyranosyl-(1?3)-O-
-arabinopyranoside] (5)
and the sugar portion see Tables 3 and 5, respectively; HRMALDI-
1b,3b,26-triol 1-O-[b-
D
a
-L
-
+
74
TOFMS [M+Na] m/z 941,4732 (calc. for C45H O19Na, 941,4722).
rhamnopyranosyl-(1?2)-O-a-L
3
.8. (25R)-26-O-b-
1b,3b,26-triol 1-O-[
glucopyranoside] (13)
D
-glucopyranosyl-22
a-methoxy-furost-5-ene-
2
D
2
Amorphous white solid; C50
H
78
O
22; ½
a
ꢂ
ꢀ25.5° (c 0.1 MeOH);
a
-
L-rhamnopyranosyl-(1?2)-O-b-D-
KBr
ꢀ1
IR
m
cm : 3429 (>OH), 2920 (>CH), 1260 and 1041 (C–O–C);
max
1
13
for H and C NMR (CD
3
OD, 600 MHz) data of the aglycon moiety
and the sugar portion see Tables 2 and 5, respectively; HRMALDI-
22
D
Amorphous white solid; C46
H
76
O
19; ½
aꢂ
ꢀ51.4° (c 0.1 MeOH);
+
TOFMS [M+Na] m/z 1053,4890 (calc. for C50
78
H O
22Na, 1053,4882).
KBr
ꢀ1
IR
m
cm : 3461 (>OH), 2933 (>CH), 1261 and 1048 (C–O–C);
max
1
13
for H and C NMR (CD
3
OD, 600 MHz) data of the aglycon moiety
and the sugar portion see Tables 3 and 5, respectively; HRMALDI-
TOFMS [M+Na] m/z 955,4885 (calc. for C46H O19Na, 955,4878).
76
3
.5. (25R)-26-O-b-
D
-glucopyranosyl-furosta-5,20(22)-diene-1b,3b,26-
+
triol 1-O-[ -rhamnopyranosyl-(1?2)-O-a-L
a-
L
-arabinopyranoside] (7)
2
D
2
3.9. (25R)-26-O-b-
D
-glucopyranosyl-furost-5-ene-1b,3b,22
a,26-tetrol
Amorphous white solid; C44
H
70
O17; ½
aꢂ
ꢀ20.3° (c 0.1 MeOH);
KBr
ꢀ1
1-O-[a
-L-rhamnopyranosyl-(1?2)-6-O-acetyl-b- -glucopyranoside]
D
IR
m
cm : 3436 (>OH), 2945 (>CH), 1272 and 1048 (C–O–C);
max
1
13
(14)
for H and C NMR (CD
3
OD, 600 MHz) data of the aglycon moiety
and the sugar portion see Tables 2 and 5, respectively; HRMALDI-
2
D
2
+
Amorphous white solid; C47
H
74
O20; ½
aꢂ
ꢀ42.9° (c 0.1 MeOH);
TOFMS [M+Na] m/z 893,4519 (calc. for C44
H
70
O
17Na, 893,4511).
KBr
ꢀ1
IR
1
m
cm : 3478 (>OH), 2938 (>CH), 1740 (C@O), 1265 and
069 (C–O–C); for H and C NMR (CD OD, 600 MHz) data of
3
max
1
13
3
2
.6. (20S)-1b,3b,16b-trihydroxypregn-5-ene-20-carboxylic acid
2,16-lactone 1-O-[ -rhamnopyranosyl-(1?4)-O-b-
the aglycon moiety and the sugar portion see Tables 3 and 5,
respectively; HRMALDITOFMS [M+Na] m/z 981,4679 (calc. for
+
a
-L
D
-
glucopyranoside] (8)
47 74
C H O20Na, 981,4671).
2
D
2
Amorphous white solid; C33
H
50
O
12; ½
a
ꢂ
ꢀ58.4° (c 0.1 MeOH);
3.10. (25R)-26-O-b-
D-glucopyranosyl-furost-5-ene-1b,3b,22a,26-
KBr
ꢀ1
IR
m
cm : 3418 (>OH), 2920 (>CH), 1745 (
and 1064 (C–O–C); for H and C NMR (CD
c
-lactone), 1259
tetrol 1-O-[ -rhamnopyranosyl-(1?2)-6-O-acetyl-b-D-
a-
L
max
1
13
3
OD, 600 MHz) data
glucopyranoside] (15)
of the aglycon moiety and the sugar portion see Tables 2 and 5,
respectively; HRMALDITOFMS [M+Na] m/z 661,3209 (calc. for
+
22
D
Amorphous white solid; C47
H
76
O
20; ½
aꢂ
ꢀ45.8° (c 0.1 MeOH);
KBr
ꢀ1
C
33
50
H O
12Na, 661,3200).
IR
m
cm : 3458 (>OH), 2947 (>CH), 1737 (C@O), 1284 and
max

A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
659
057 (C–O–C); for H and 13C NMR (CD
1
3.13. (25R)-26-O-b-
D-glucopyranosyl-furost-5-ene-3b,22a,26-triol 3-
1
3
OD, 600 MHz) data of the
aglycon moiety and the sugar portion see Tables 3 and 5,
respectively; HRMALDITOFMS [M+Na] m/z 983,4834 (calc. for
O-{ -arabinopyranosyl-(1?4)-O-b- -glucopyranoside (18)
a
-
L
D
+
2
2
C
47
H
76
O20Na, 983,4828).
Amorphous white solid; C44
H
72
O18; ½
a
ꢂ
–67.2° (c 0.1 MeOH);
D
KBr
ꢀ1
IR
m
cm : 3451 (>OH), 2930 (>CH), 1275 and 1040 (C–O–C);
max
1
13
for H and C NMR (CD
3
OD, 600 MHz) data of the aglycon moiety
3
1
.11. (25R)-26-O-b-
b,3b,26-triol 1-O-[
D
-glucopyranosyl-22
a
-methoxy-furost-5-ene-
and the sugar portion see Tables 4 and 5, respectively; HRMALDI-
TOFMS [M+Na] m/z 911,4625 (calc. for C44H O18Na, 911,4616).
72
+
a
-
L
-rhamnopyranosyl-(1?2)-6-O-acetyl-b-
D
-
glucopyranoside] (16)
2
D
2
3.14. (25R)-26-O-b-
b,3b,26-triol 1-O-[
glucopyranoside] (19)
D-glucopyranosyl-furosta-5,20(22)-diene-
Amorphous white solid; C48
H
78
O
20; ½
a
ꢂ
ꢀ57.3° (c 0.1 MeOH);
1
a-
L-rhamnopyranosyl-(1?2)-6-O-acetyl-b-D-
KBr
ꢀ1
IR
1
m
cm : 3480 (>OH), 2946 (>CH), 1732 (C@O), 1278 and
062 (C–O–C); for H and C NMR (CD OD, 600 MHz) data of
3
max
1
13
the aglycon moiety and the sugar portion see Tables 3 and 5,
respectively; HRMALDITOFMS [M+H] m/z 997,4992 (calc. for
C H O20Na, 997,4984).
48 78
22
D
Amorphous white solid; C47
H
74
O
19; ½
a
ꢂ
ꢀ31.0° (c 0.1 MeOH);
+
KBr
ꢀ1
IR
1
m
cm : 3444 (>OH), 2938 (>CH), 1735 (C@O), 1277 and
054 (C–O–C); for H and C NMR (CD OD, 600 MHz) data of
3
max
1
13
the aglycon moiety and the sugar portion see Tables 2 and 5,
+
respectively; HRMALDITOFMS [M+Na] m/z 965,4731 (calc. for
C H O19Na, 965,4722).
45 74
3
.12. 26-O-b-
D
-glucopyranosyl-furosta-5,25(27)-diene-3b,22
a,26-
triol 3-O-[ -arabinopyranosyl-(1?4)-O-b-D
a-L
-glucopyranoside] (17)
2
D
2
Amorphous white solid; C44
H
70
O
18; ½
a
ꢂ
ꢀ72.6° (c 0.1 MeOH);
3.15. (25R)-26-O-b-
D
-glucopyranosyl-furosta-5,20(22)-diene-3b,26-
KBr
ꢀ1
IR
m
cm : 3463 (>OH), 2941 (>CH), 1264 and 1062 (C–O–C);
for H and C NMR (CD OD, 600 MHz) data of the aglycon moiety
3
and the sugar portion see Tables 4 and 5, respectively; HRMALDI-
diol 3-O-[ -arabinopyranosyl-(1?4)-O-b-D
a-
L
-glucopyranoside] (20)
max
1
13
2
D
2
Amorphous white solid; C44
70
H O
17; ½
aꢂ
ꢀ55.0° (c 0.1 MeOH);
+
KBr
max
ꢀ1
TOFMS [M+Na] m/z 909,4467 (calc. for C44
H
70
O18Na, 909,4460).
IR
m
cm : 3468 (>OH), 2953 (>CH), 1280 and 1043 (C–O–C);
Table 5
1
3C and 1H NMR data (J in Hz) of the sugar portions of compounds 5, 7, 12, 18 and 19 (600 MHz, CD
3
OD).
5
7a
12a
18b
19c
a-
L-Ara
a
-
L-Ara
b-
D-GlcI
b-
D-GlcI
6-O-Ac-b-D-GlcI
1
2
3
4
5
100.9 4.30 d (3.7)
75.3 3.73 dd (8.5, 3.7)
75.9 3.67 dd (8.5, 3.0)
70.6 3.77 m
100.6 4.30 d (3.7)
75.2 3.73 dd (8.5, 3.7)
75.7 3.67 dd (8.5, 3.0)
70.5 3.76 m
67.1 3.87 dd (11.9, 2.0)
3.52 dd (11.9, 3.0)
100.0 4.40 d (7.5)
101.8 4.45 d (7.5)
74.6 3.24 dd (9.0, 7.5)
76.0 3.55 dd (9.0, 9.0)
80.4 3.57 dd (9.0, 9.0)
76.3 3.45 ddd (9.0, 4.5,
2.0)
100.5 4.38 d (7.5)
77.2 3.44 dd (9.0, 7.5)
79.6 3.51 dd (9.0, 9.0)
72.2 3.19 dd (9.0, 9.0)
74.2 3.45 ddd (9.0, 4.5,
2.0)
77.3 3.44 dd (9.0, 7.5)
79.6 3.50 dd (9.0, 9.0)
71.8 3.32 dd (9.0, 9.0)
77.6 3.27 ddd (9.0, 4.5,
2.0)
67.3 3.88 dd (11.9, 2.0)
3.54 dd (11.9, 3.0)
6
63.4 3.93 dd (12.0, 2.0)
61.2 3.90 dd (12.0, 2.0)
3.86 dd (12.0, 4.5)
65.0 4.35 dd (12.0, 2.1)
4.31 dd (12.0, 4.5)
3.63 dd (12.0, 4.5)
OCOCH
OCOCH
3
172.3
20.8 2.08 s
-Rha
–
3
a-
L-Rha
a
-
L-Rha
a
-
L-Rha
a-
L
-Ara
a-L
1
2
3
4
5
101.2 5.33 d (1.2)
71.2 4.19 dd (3.2, 1.2)
82.6 3.84 dd (9.3, 3.2)
72.4 3.61 t (9.3)
101.2 5.33 d (1.2)
72.1 3.91 dd (3.2, 1.2)
71.8 3.72 dd (9.3, 3.2)
73.8 3.43 t (9.3)
101.0 5.37 d (1.2)
72.0 3.91 dd (3.2, 1.2)
71.8 3.71 dd (9.3, 3.2)
73.8 3.42 t (9.3)
105.1 4.33 d (3.7)
72.1 3.59 dd (8.5, 3.7)
74.1 3.55 dd (8.5, 3.0)
69.8 3.85 m
101.1 5.37 d (1.2)
72.1 3.92 dd (3.2, 1.2)
71.9 3.72 dd (9.3, 3.2)
73.9 3.43 t (9.3)
69.4 4.13 m
69.0 4.18 m
69.3 4.11 m
69.3 4.12 m
67.3 3.96 dd (11.9, 2.0)
3.66 dd (11.9, 3.0)
6
18.1 1.29 d (6.0)
18.2 1.28 d (6.0)
18.2 1.27 d (6.0)
18.0 1.28 d (6.0)
b-
D
-GlcI
b-
D-Glc
b-
D-GlcII
b-D-GlcII
b-D-GlcII
1
2
3
4
5
105.6 4.57 d (7.5)
104.4 4.27 d (7.5)
74.9 3.22 dd (9.0, 7.5)
77.7 3.37 dd (9.0, 9.0)
71.5 3.31 dd (9.0, 9.0)
77.7 3.29 ddd (9.0, 4.5,
2.0)
104.3 4.27 d (7.5)
74.8 3.22 dd (9.0, 7.5)
77.7 3.38 dd (9.0, 9.0)
71.3 3.31 dd (9.0, 9.0)
77.6 3.30 ddd (9.0, 4.5,
2.0)
104.3 4.27 d (7.5)
74.6 3.23 dd (9.0, 7.5)
77.7 3.38 dd (9.0, 9.0)
71.3 3.31 dd (9.0, 9.0)
77.6 3.29 ddd (9.0, 4.5,
2.0)
104.2 4.28 d (7.5)
74.9 3.22 dd (9.0, 7.5)
77.8 3.39 dd (9.0, 9.0)
71.4 3.32 dd (9.0, 9.0)
77.5 3.30 ddd (9.0, 4.5,
2.0)
75.1 3.33 dd (9.0, 7.5)
77.8 3.40 dd (9.0, 9.0)
71.0 3.38 dd (9.0, 9.0)
77.8 3.28 ddd (9.0, 4.5,
2
.0)
62.1 3.88 dd (12.0, 2.0)
.73 dd (12.0, 4.5)
b- -GlcII
6
62.6 3.90 dd (12.0, 2.0)
3.69 dd (12.0, 4.5)
62.4 3.89 dd (12.0, 2.0)
3.69 dd (12.0, 4.5)
62.3 3.89 dd (12.0, 2.0)
3.70 dd (12.0, 4.5)
62.7 3.90 dd (12.0, 2.0)
3.70 dd (12.0, 4.5)
3
D
1
2
3
4
5
102.9 4.31 d (7.5)
74.9 3.24 dd (9.0, 7.5)
77.8 3.37 dd (9.0, 9.0)
71.3 3.32 dd (9.0, 9.0)
77.8 3.28 ddd (9.0, 4.5,
2
.0)
62.6 3.91 dd (12.0, 2.0)
.69 dd (12.0, 4.5)
6
3
a
b
c
The chemical shift values of the sugar portion of 8 and 13 deviate from the experimental values of 7 and 12 respectively, of ±0.03 ppm.
The chemical shift values of the sugar portion of 17 and 20 deviate from the experimental values of 18 of ±0.03 ppm.
The chemical shift values of the sugar portion of 14–16 deviate from the experimental values of 19 of ±0.03 ppm.

6
60
A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
OH α-L-ara
O
HO
O
5
7
R1 =
R2 = CH2
α-L-rha
O
HO
O
OH
O
HO
HO
OH
OH
β-D-glc
2
R2
7
2
1
α-L-ara
O
OH
HO
2
0
O
2
5
2
2
OR1
R =
R2 = (25R)-CH3
1
O
OH
O
O
HO
HO
α-L-rha
O
HO
OH
HO
HO
β-D-glc
OH
β-D-glc
OCOCH3
O
HO
HO
1
9
R =
O
R2 = (25R)-CH3
1
α-L-rha
HO
HO
O
OH
O
α-L-ara
O
O
OH
HO
O
OH
O
O
O
β-D-glc
α-L-ara
HO
HO
O
OH
HO
OH
O
O
OH
O
O
β-D-glc
HO
O
HO
HO
OH
OH
OH
HO
α-L-rha
8
20
1
2
R1 = H
R1 = H
R2 = H
R3 = (25R)-CH3
R3 = (25R)-CH3
β-D-glc
OR1
R3
OR2
O
1
3
R2 = CH3
R2 = H
O
O
HO
HO
OH
O
O
O
14
15
R1 = COCH3
R1 = COCH3
R1 = COCH3
R3 = CH2
HO
HO
HO
O
β-D-glc OH
2
R = H
R3 = (25R)-CH3
R3 = (25R)-CH3
HO
OH
α-L-rha
HO
16
R2 = CH3
R2
O
OR1
O
1
7
R1 = H
R1 = H
R2 = CH2
O
OH
β-D-glc
18
R2 = (25R)-CH3
α-L-ara
HO
HO
OH
HO
OH
O
O
β-D-glc OH
O
O
HO
OH
OH
for H and 13C NMR (CD
1
OD, 600 MHz) data of the aglycon moiety
3
3 2
using CHCl –MeOH–H O (7:1:1.2, lower layer) as eluting solvent
and the sugar portion see Tables 4 and 5, respectively; HRMALDI-
to afford glucose, arabinose and rhamnose.
The configuration glucose and the
+
TOFMS [M+Na] m/z 893,4517 (calc. for C44
H
70
O
17Na, 893,4511).
D
L
configuration of
rhamnose and arabinose were established as by comparison
of their optical rotation values with those reported in the lit-
3
.16. Acid hydrolysis
23
D
23
D
erature:
et al., 2008),
optical rotations were determined after dissolving the sugars
and allowing them to stand for 24 h: -glucose
-rhamnose
D
-glucose
½
a
ꢂ
+ 52.5,
L
-rhamnose
½
a
ꢂ
ꢀ 4.4 (Wang
2
3
L
-arabinose ½
a
ꢂ
+ 105.0 (Belitz et al., 2009). The
D
The crude saponin mixture (1 g) was heated at 60 °C with 1:1
.5 N HCl-dioxane (100 ml) for 2 h, and the mixture was then evap-
0
in 23H
2
O
D
orated in vacuo. The residue was partitioned with CH
the H O layer was neutralized with Amberlite MB-3. The H
was then concentrated and passed through a silica gel column,
2
Cl
2
–H
2
O, and
O layer
23
½
½
a
ꢂ
ꢂ
+ 53.4 (c 0.1),
ꢀ 4.9 (c 0.1).
L-arabinose ½
aꢂ
+ 106.2 (c 0.1), L
D
D
23
D
2
2
a

A. Napolitano et al. / Phytochemistry 72 (2011) 651–661
661
3
.17. Enzymatic hydrolysis of compound 7
Kereselidze, E.V., Pkheidze, T.A., Kemertelidze, E.P., Khardziani, S.D., Dzhaparidze,
T.N., Makharadze, Sh.K., 1975. Fibrinolytic activity of Ruscus ponticus and Ruscus
hypophyllum saponins. Soobsch. AN GSSR 78, 485–488.
Korkashvili, T.Sh., Dzhikiya, O.D., Vugalter, M.M., Pkheidze, T.A., Kemertelidze, E.P.,
1985. Steroid glycosides of Ruscus ponticus. Soobsch. AN GSSR 120, 561–564.
Liu, H.Y., Chen, C.X., 2002. Two new steroidal saponins from Tacca plantaginea. Chin.
Chem. Lett. 13, 633–636.
Mimaki, Y., Takaashi, Y., Kuroda, M., Sashida, Y., Nikaido, T., 1996. Steroidal saponins
from Nolina recurvata stems and their inhibitory activity on cyclic AMP
phosphodiesterase. Phytochemistry 42, 1609–1615.
Mimaki, Y., Kuroda, M., Kameyama, A., Yokosuka, A., Sashida, Y., 1998. New steroidal
constituents of the underground parts of Ruscus aculeatus and their cytostatic
activity on HL-60 cells. Chem. Pharm. Bull. 46, 298–303.
Compounds 7 (2.8 mg) was treated with 10 mg of b-glucosidase
and 5 ml of 0.5% phosphoric acid at 37 °C for 24 h. After cooling,
each solution was extracted three times with n-BuOH. The n-BuOH
layers were concentrated and dried with a N draft affording the
2
corresponding spirostanol glycosides characterized by NMR
analysis.
The NMR data of the aglycon moiety of the spirostanol deriva-
tive of compound 7 were in agreement with those reported for
Mulkijanyan, A.K., Abuladze, G., 1998. Antiinflamatory activity of the steroidal
saponins of Ruscus ponticus. Proc. Georg. Acad. Sci. 24 (1–6), 265–269.
Mulkijanyan, K., Abuladze, G., 2000. Antiexudative action of steroid glycosides’
preparation ruscoponin from Ruscus ponticus. Bull. Georg. Acad. Sci. 161, 254–
(
1
25R)-spirost-5-ene-1b,3b-diol or ruscogenin (Agrawal et al.,
985).
256.
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