Iridoid glycosides from the leaves of Sambucus ebulus

Article abstract of DOI:10.1021/np900373u

Six new iridoid glycosides (1-6) of the "Valeriana type" were isolated from leaves of Sambucus ebulus. The structures were elucidated by 1D- and 2D-NMR spectroscopy, mass spectrometry, and chemical degradation methods as 10-O-acetylpatrinoside-aglycone-11-O-[4″-O-acetyl-α-L- rhamnopyranosyl-(1→2)-β-D-ribohexo-3-ulopyranoside] (1), 7-O-acetylpatrinoside-aglycone-11-O-[4″-O-acetyl-α-L- rhamnopyranosyl-(1→2)-β-D-ribohexo-3-ulopyranoside] (2), 10-O-acetylpatrinoside-aglycone-11-O-[α-L-rhamnopyranosyl-(1→2) -β-D-ribohexo-3-ulopyranoside] (3), patrinoside-aglycone-11-O-[4″-O- acetyl-α-L-rhamnopyranosyl-(1→2)-β-D-ribohexo-3-ulopyranoside] (4), 10-O-acetylpatrinoside-aglycone-11-O-[4″-O-acetyl-α-L- rhamnopyranosyl-(1→2)-β-D-glucopyranoside] (5), and patrinoside-aglycone-11-O-2′-deoxy-β-D-glucopyranoside (6). Compounds 1-4 represent the first examples of acylated iridoid diglycosides bearing the uncommon D-ribohexo-3-ulopyranosyl sugar moiety. Compound 6 is the first iridoid glycoside with a 2-deoxy-D-glucopyranosyl sugar moiety.

Full text of DOI:10.1021/np900373u

1798
J. Nat. Prod. 2009, 72, 1798–1803
Iridoid Glycosides from the Leaves of Sambucus ebulus
Valerio Pieri,†,§ Stefan Schwaiger,†,§ Ernst P. Ellmerer,‡ and Hermann Stuppner*,†,§
Institute of Pharmacy/Pharmacognosy, UniVersity of Innsbruck, Josef-Moeller-Haus, Innrain 52c, 6020 Innsbruck, Austria, Institute of Organic
Chemistry, UniVersity of Innsbruck, Innrain 52a, 6020 Innsbruck, Austria, and Center for Molecular Biosciences Innsbruck, Austria
ReceiVed June 19, 2009
Six new iridoid glycosides (1-6) of the “Valeriana type” were isolated from leaves of Sambucus ebulus. The structures
were elucidated by 1D- and 2D-NMR spectroscopy, mass spectrometry, and chemical degradation methods as
10-O-acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (1), 7-O-
acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (2), 10-O-
acetylpatrinoside-aglycone-11-O-[R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (3), patrinoside-aglycone-
11-O-[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (4), 10-O-acetylpatrinoside-aglycone-
11-O-[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-glucopyranoside] (5), and patrinoside-aglycone-11-O-2′-deoxy-ꢀ-
D-glucopyranoside (6). Compounds 1-4 represent the first examples of acylated iridoid diglycosides bearing the
uncommon D-ribohexo-3-ulopyranosyl sugar moiety. Compound 6 is the first iridoid glycoside with a 2-deoxy-D-
glucopyranosyl sugar moiety.
Sambucus ebulus L. (Adoxaceae), commonly referred to as
“dwarf elder”, is a perennial herbaceous plant distributed in most
of Europe, northern Africa, and western Asia.1 Various parts of
the plant are used in traditional medicine as a remedy for several
pathological processes related to inflammation and as a diuretic.1,2
Extracts obtained from the plant have shown anti-inflammatory,2-6
antinociceptive,2 diuretic,7 hypotensive,8 anti-Helicobacter pylori,9
and antioxidative10,11 properties.
Hitherto, phytochemical investigations have been focused on the
root material of S. ebulus, resulting in the isolation of four iridoid
glycosides, namely, ebuloside,12 7,7-O-dihydroebuloside,13 6′-O-
apiosylebuloside,13 and morroniside,14 together with the secoiridoid
glycoside isosweroside.14 Despite wide use of the leaves as a
traditional remedy, there have been no reports on the isolation of
secondary metabolites from the leaves of S. ebulus. This study deals
with the isolation and structure elucidation of six new iridoid
glycosides of the “Valeriana type” (1-6) from leaves of S. ebulus.
Results and Discussion
Air-dried leaves of S. ebulus were ground and extracted with
EtOH. Removal of the solvent under reduced pressure yielded a
crude extract, which was suspended in H2O and sequentially
partitioned with petroleum ether, diethyl ether, EtOAc, and n-BuOH.
The EtOAc solubles were fractionated by a combination of column
chromatography (CC), high-speed counter-current chromatography
(HSCCC), and semipreparative HPLC to yield compounds 1-6.
Compound 1 was isolated as a white, amorphous solid. The
HRFABMS spectrum displayed a quasimolecular ion at m/z 691.28
([M + H]+), consistent with the chemical formula C31H46O17.
Analysis of the 1H, HSQC, and HMBC NMR spectra revealed the
presence of five methyl, five methylene, 16 methine, and five
quaternary carbons. In the 13C NMR spectrum, resonances of an
isovaleryl moiety (δ 22.4, 25.9, 43.4, 172.0), two acetyl residues
(δ 20.9, 171.0; δ 21.3, 171.0), two anomeric carbons (δ 100.3,
103.0), and a ketonic function (δ 206.3) were observed (Table 1).
1
The downfield-shifted H NMR signals at δ 6.51 (d, J ) 4.9 Hz)
and 6.62 (br s) were assigned to H-1 and H-3 of an iridoid backbone,
respectively. Extensive analysis of the COSY spectrum enabled the
unambiguous assignment of four further methine (CH-5, CH-7, CH-
8, CH-9) and three methylene groups (CH2-6, CH2-10, CH2-11) to
the iridoid backbone. HMBC correlations between H-1 (δ 6.51)
and the carbonyl carbon of the isovaleryl group (δ 172.0) and
between H2-10 (δa 4.83, δb 4.68) and the carbonyl carbon of one
acetyl residue (δ 171.0) indicated esterification of O-1 and O-10
* To whom correspondence should be addressed. Tel: +43-512-507-
5300. Fax: +43-512-507-2939. E-mail: Hermann.Stuppner@uibk.ac.at.
† Institute of Pharmacy/Pharmacognosy, University of Innsbruck.
‡ Institute of Organic Chemistry, University of Innsbruck.
§ Center for Molecular Biosciences Innsbruck.
10.1021/np900373u CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/01/2009

Iridoid Glycosides from Sambucus ebulus
Journal of Natural Products, 2009, Vol. 72, No. 10 1799
Table 1. 1H and 13C NMR Data for Compounds 1-3 (C5D5N)
1
2
3
b
b
b
position
δH (J in Hz)a
δC
δH (J in Hz)a
δC
δH (J in Hz)a
δC
aglycon
1
3
4
5
6R
6ꢀ
7
8
9
10a
10b
11a
11b
6.51, d (4.9)
6.62, br s
92.4
139.8
115.3
33.3
6.78, d (4.0)
6.57, br s
92.0
139.9
114.2
32.2
6.34, d (6.0)
6.56, br s
93.2
139.7
115.9
33.5
3.48, q-like
3.40, q-like
2.32, mc
3.61, q-like
1.90, m
2.14, mc
40.7
36.7
41.1
2.52, ddd (13.2, 7.3, 3.0)
2.40, mc
2.56, ddd (13.2, 7.2, 2.5)
4.67, mc
71.0
45.6
42.8
64.5
5.70, q-like
2.64, m
2.83, td (8.7, 4.0)
4.21, dd (10.6, 6.9)
4.11, dd (10.6, 6.9)
4.49, d (11.2)
75.2
46.6
43.3
61.1
4.61, mc
71.2
46.1
42.6
64.3
2.42, m
2.38, mc
2.67, td (8.7, 4.9)
4.83, dd (10.9, 8.0)
4.68, mc
4.52, d (11.1)
4.28, d (11.1)
2.61, td (8.4, 6.0)
4.76, dd (10.7, 8.7)
4.64, dd (10.7, 6.5)
4.52, d (11.1)
4.24, d (11.1)
69.7
69.5
69.4
4.25, d (11.2)
isovaleryl
-COO-
-CH2-
-CH<
(-CH3)a
(-CH3)b
sugar
1′
172.0
43.4
25.9
22.4
171.7
43.4
25.8
22.3
172.0
43.4
25.9
22.5
2.36, m
2.30, m
2.37, mc
2.24 - 2.13, m
0.94, d (6.8)
0.93, d (6.8)
2.23-2.15, mc
0.95, d (6.8)
0.93, d (6.8)
2.20 - 2.10, mc
0.91, d (6.7)
0.91, d (6.7)
4.95, d (8.0)
4.92, dd (8.0, 1.4)
103.0
79.2
206.3
74.0
78.6
62.2
4.95, mc,d
4.92, mc,d
103.1
79.1
206.2
74.0
78.6
62.2
4.94, d (8.0)
4.97, dd (8.0, 1.5)
103.0
79.1
206.7
74.1
78.6
62.2
2′
3′
4′
4.93, dd (10.1, 1.4)
3.87, ddd (10.1, 4.3, 1.9)
4.51, dd (12.1, 1.9)
4.41, dd (12.1, 4.3)
5.78, d (1.3)
4.92, mc
4.93, dd (10.0, 1.5)
3.86, ddd (10.0, 4.4, 2.0)
4.51, dd (12.2, 2.0)
4.41, dd (12.2, 4.4)
5.82, d (1.3)
5′
3.88, ddd (10.1, 4.3, 1.9)
4.52, dd (12.1, 1.9)
4.41, dd (12.1, 4.3)
5.78, d (1.3)
4.85, dd (3.4, 1.3)
4.56, dd (9.8, 3.4)
5.83, t (9.8)
6′a
6′b
1′′
100.3
71.8
70.0
75.7
67.5
18.1
100.3
71.8
70.1
75.6
67.4
18.0
100.5
72.0
72.4
74.1
70.0
18.6
2′′
4.86, dd (3.4, 1.3)
4.88, dd (3.4, 1.3)
3′′
4.64, mc
4.58, mc
4′′
5.84, t (9.8)
4.29, t (9.5)
5′′
4.65, mc
4.62, dq (9.8, 6.3)
1.48, d (6.3)
4.58, mc
6′′
1.48, d (6.3)
1.65, d (6.2)
acetyl
7-COO-
7-CH3
10-COO-
10-CH3
4′′-COO-
4′′-CH3
170.7e
21.0f
2.08, se
171.0e
20.9f
170.9
21.0
2.02, se
2.21, se
2.00, s
171.0e
21.3f
170.8e
21.3f
2.22, se
a Recorded at 600 MHz. b Assignments based on 2D HSQC and HMBC spectra recorded at 75 MHz. c Overlapping signals. d Signal resolved in the
1H NMR spectrum measured in DMSO-d6 (Table 4, Supporting Information). e,f Signals in the same column may be interchanged.
20
with isovaleric and acetic acid, respectively. A further HMBC
correlation was found between H2-11 (δa 4.52, δb 4.28) and the
anomeric carbon (δ 103.0) of a sugar moiety, showing glycosylation
of O-11. The 1H and 13C NMR data of the sugar residue (Tables 1
and 3) were in good agreement with those reported for D-ribohexo-
3-ulopyranose.15-18 In particular, the COSY and HMBC NMR
spectra showed correlations identical to those described for the sugar
moiety of 1H-indol-3-yl-ꢀ-D-ribohex-3′-ulopyranoside18 and were
consistent with the presence of a keto group in position 3′. Briefly,
COSY correlations were found between H-1′ and H-2′, H2-6′ and
H-5′, and H-5′ and H-4′. HMBC correlations were found between
C-3′ and H-1′, H-2′, H-4′, and H-5′. The ꢀ glycosidic linkage of
the sugar moiety was deduced from the coupling constant J1′, 2′ (8.0
Hz).18 Further signals were assigned to a rhamnopyranosyl residue
on the basis of comparison with 13C and 1H NMR literature data19
and by extensive analysis of the COSY spectrum. The HMBC
correlation between H-4′′ (δ 5.84) of the rhamnopyranosyl moiety
and the carbonyl carbon (δ 171.0) of an acetyl residue indicated
acetylation of the rhamnopyranosyl residue at O-4′′. The HMBC
spectrum also revealed a cross-peak between the anomeric proton
of 4′′-O-acetylrhamnopyranose (δ 5.78) and C-2′ (δ 79.2) of
ribohexo-3-ulopyranose, thus indicating a 4′′-O-acetylrhamnopy-
ranosyl-(1f2)-ribohexo-3-ulopyranosyl linkage. The coupling con-
stant J1′′, 2′′ ) 1.3 Hz suggested an R glycosidic linkage of the 4′′-
O-acetylrhamnopyranosyl residue.19 Acid hydrolysis followed by
reduction with NaBH4 provided D-allitol and D-sorbitol as
determined by GC analysis and thus proved the absolute config-
uration of D-ribohexo-3-ulose. The absolute configuration of L-
rhamnose was confirmed by hydrolysis followed by GC analysis
of its thiazolidine derivative.21 The relative configurations of C-1,
C-5, C-7, C-8, and C-9 of the aglycon part of the molecule were
1
established on the basis of H and ROESY NMR spectra. The
coupling constants J1, 9 (4.9 Hz) and J5, 9 (8.7 Hz) were consistent
with H-1R, H-5ꢀ, and H-9ꢀ orientations. The ROESY spectrum
showed ROE correlations between H-1 (δ 6.51) and H-8 (δ 2.42),
H-9 (δ 2.67), and H2-10 (δa 4.83, δb 4.68) and thus indicated the
R orientation of H-8. Furthermore, a strong correlation was observed
between H-7 (δ 4.67) and H-8, which, however, was not conclusive
for the R orientation of H-7, as NOE contacts between vicinal
protons of an iridoid backbone with a trans-relationship may be
observed.22 Therefore, in order to determine the orientation of H-7,
the ROE contacts between H-7 and H2-6 (δR 2.14, δꢀ 2.52) were
considered. In particular, a stronger correlation between H-7 and
H-6R compared to the correlation between H-7 and H-6ꢀ was found,
1
which indicated the R orientation of H-7. The validity of the H
NMR signal assignments for H-6R and H-6ꢀ was proved by
observing the ROE contacts between H-5 (δ 3.48) and H-6ꢀ, H-5,
and H-6R, which respectively showed strong and weak correlations.
The R orientation of H-7 was also supported by the absence of
interactions between H-7 and H-5 and between H-7 and H-9. Thus,

1800 Journal of Natural Products, 2009, Vol. 72, No. 10
Table 2. 1H and 13C NMR Data for Compounds 4-6 (C5D5N)
Pieri et al.
4
5
6
b
b
b
position
δH (J in Hz)a
δC
δH (J in Hz)a
δC
δH (J in Hz)a
δC
aglycon
1
3
4
5
6R
6ꢀ
7
8
9
10a
10b
11a
11b
6.68, d (4.5)
6.59, br s
92.6
139.7
115.0
33.0
6.49, d (4.9)
6.63, br s
92.5
139.7
115.6
33.5
6.552, d (5.3)
6.560, br s
93.0
138.9
116.0
33.8
3.49, q-like
3.49, q-like
2.14, mc
3.39, q-like
2.00, m
2.43, m (13.0, 7.3, 3.1)
4.76, q-like
2.22, mc
40.6
40.8
41.2
2.51, ddd (13.1, 7.3, 3.9)
4.81, q-like
2.42, dq (8.7, 6.1)
2.91, td (8.7, 4.5)
4.41, mc
4.33, dd (10.8, 5.6)
4.52, d (11.1)
4.26, d (11.1)
2.54, ddd (13.2, 7.1, 2.9)
72.7
48.1
42.3
62.0
4.65, mc
71.0
45.7
42.9
64.5
72.8
48.7
42.3
62.1
2.41, m
2.39, m
2.65, td (8.6, 4.9)
4.81, dd (10.9, 8.1)
4.67, mc
2.76, td (8.6, 5.3)
4.37, mc
4.30, dd (10.8, 5.4)
4.46, d (11.7)
4.20, d (11.7)
69.8
4.53, mc
69.2
68.6
4.25, mc
isovaleryl
-COO-
-CH2-
-CH<
(-CH3)a
(-CH3)b
sugar
1′
171.9
43.4
25.9
22.4
172.0
43.4
25.9
22.4
171.9
43.4
25.8
22.4
2.29, m
2.36, m
2.23, m
2.18-2.09, mc
0.910, d (6.7)
0.909, d (6.7)
2.22-2.12, mc
0.93, d (6.8)
0.92, d (6.8)
2.18-2.05, mc
0.88, d (6.7)
0.88, d (6.7)
4.95, mc,d
4.91, mc,d
103.0
79.1
4.85, mc,d
4.23, mc,d
102.4
77.5
4.90, dd (9.7, 1.8)
2.56, ddd (12.3, 4.8, 1.8)
2.11, mc
4.18, ddd (11.8, 8.6, 4.8)
4.04, t (9.0)
3.81, ddd (9.4, 5.2, 2.4)
4.54, dd (11.8, 2.4)
4.39, mc
100.1
40.8
2′a
2′b
3′
206.2
74.0
78.6
62.2
4.24, mc
4.12, t (9.0)
3.88, ddd (9.4, 5.4, 2.2)
4.51, mc
4.32, dd (11.9, 5.4)
6.42, d (1.3)
4.75, m (3.4, 1.3)
4.66, mc
79.7
71.9
78.5
62.7
72.4
73.5
78.7
63.0
4′
4.93, mc
5′
3.87, ddd (10.1, 4.0, 1.8)
6′a
4.51, mc
6′b
4.41, mc
1′′
5.78, d (1.3)
100.3
71.9
70.0
75.7
67.5
18.1
101.6
72.5
70.4
76.1
67.0
18.3
2′′
4.87, mc
3′′
4.64, mc
4′′
5.85, t (9.8)
5.87, t (9.8)
5′′
4.66, mc
4.84, mc
6′′
1.48, d (6.3)
1.53, d (6.2)
acetyl
10-COO-
10-CH3
4′′-COO-
4′′-CH3
171.0e
21.0f
2.01, se
2.21, se
171.0
21.3
171.0e
2.21, s
21.3f
a Recorded at 600 MHz. b Assignments based on 2D HSQC and HMBC spectra recorded at 75 MHz. c Overlapping signals. d Signal resolved in the
1H NMR spectrum measured in DMSO-d6 (Tables 3 and 4, Supporting Information). e,f Signals in the same column may be interchanged.
the configurations of C-1, C-5, C-7, C-8, and C-9 were proved to
be identical to those of patrinoside.23 The structure of 1 was
therefore elucidated as 10-O-acetylpatrinoside-aglycone-11-O-[4′′-
O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyrano-
side].
Compound 3 was isolated as a white, amorphous solid. The
HRFABMS spectrum displayed a quasimolecular ion at m/z 649.27
([M + H]+), consistent with the chemical formula C29H44O16 and
thus suggesting the lack of one acetyl group compared to 1. As
expected, comparison of 13C and 1H spectroscopic data of 3 and 1
showed the lack of one methyl group and one quaternary carbon.
Moreover, it was found that the signal of H′′-4 of the rhamnopy-
ranosyl residue was shifted upfield (δ 4.29) compared to 1 (δ 5.84),
thus indicating the presence of a free OH group at this position.
No correlation between H′′-4 and quaternary carbons was observed
in the HMBC spectrum. The ꢀ glycosidic linkage of the ribohexo-
3-ulopyranosyl sugar moiety and the R glycosidic linkage of the
rhamnopyranosyl residue were deduced from the anomeric coupling
constants (J1′, 2′ ) 8.0 Hz and J1′′, 2′′ ) 1.3 Hz, respectively).18,19
Acid hydrolysis followed by derivatization and GC analysis
provided results identical to those of compound 1. The 13C and 1H
spectroscopic data for the aglycon part of 3 were almost identical
to those of 1, indicating that the two compounds possessed the same
aglycon. The relative configurations of C-1, C-5, C-7, C-8, and
C-9 were also found to be identical to those of 1, as deduced from
1H and ROESY NMR data. Therefore, the structure of 3 was
elucidated as 10-O-acetylpatrinoside-aglycone-11-O-[R-L-rham-
nopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside].
Compound 2 was isolated as a white, amorphous solid. The
HRFABMS spectrum displayed a quasimolecular ion at m/z 691.28
([M + H]+), consistent with the chemical formula C31H46O17.
Compounds 1 and 2 were thus isomers. Comparison of 13C and 1H
spectroscopic data of compounds 1 and 2 showed almost identical
signals for the sugar moieties, suggesting differences only in the
aglycon part of the molecules. In particular, the downfield values
for CH-7 (δC 75.2, δH 5.70) and the upfield values for CH2-10 (δC
61.1, δH 4.21, 4.11) compared to those of 1 indicated the presence
of an acetoxy group at CH-7 and a free OH group at CH2-10.
Analysis of the HMBC spectrum showed the expected correlation
between H-7 (δ 5.70) and the quaternary carbon of the acetyl group
(δ 170.7). The relative configurations of C-1, C-5, C-7, C-8, and
C-9 of the aglycon part were deduced on the basis of 1H and
ROESY NMR spectra and were found to be identical to those of
1. Acid hydrolysis followed by derivatization and GC analysis
confirmed NMR signal assignments in Table 1. Therefore, the
structure of 2 was elucidated as 7-O-acetylpatrinoside-aglycone-
11-O-[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ul-
opyranoside].
Compound 4 was isolated as a light yellow, amorphous solid.
The HRFABMS spectrum displayed a quasimolecular ion at m/z

Iridoid Glycosides from Sambucus ebulus
Journal of Natural Products, 2009, Vol. 72, No. 10 1801
Table 3. 1H and 13C NMR Data for Compounds 1 and 5
(DMSO-d6)
sugar moiety were assigned to glucopyranose, which was in
complete agreement with literature data.16,17,24 The glycosylation
position was determined to be at C-11 of the aglycon by analysis
of the HMBC spectrum, which showed a correlation between H2-
11 (δ 4.53, 4.25) and C-1′ (δ 102.4) of glucopyranose. A further
HMBC correlation between the anomeric proton of 4′′-O-acetyl-
rhamnopyranose (δ 6.42) and C-2′ (δ 77.5) of the glucopyranosyl
residue confirmed the 4′′-O-acetylrhamnopyranosyl-(1f2)-glu-
copyranosyl linkage. The coupling constants J1′,2′ ) 7.9 Hz and
J1′′,2′′ ) 1.3 Hz indicated a ꢀ glycosidic linkage of the glucopyra-
nosyl sugar moiety and an R glycosidic linkage of the 4′′-O-
acetylrhamnopyranosyl residue.16,19 Acid hydrolysis followed by
derivatization with L-cysteine methyl ester hydrochloride confirmed
the absolute configurations of the sugar moieties as D-glucose and
L-rhamnose as determined by GC analysis. Thus, 5 is 10-O-
acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-R-L-rhamnopyrano-
syl-(1f2)-ꢀ-D-glucopyranoside].
1
5
b
b
position
δH (J in Hz)a
δC
δH (J in Hz)a
δC
aglycon
1
3
4
5
6R
6ꢀ
7
8
9
10a
5.93, d (4.5)
6.40, br s
90.6 5.90, d (4.6)
138.1 6.36, br s
113.8
90.7
138.1
113.7
31.5
2.85, q-like
1.81, m
31.3 2.85, q-like
38.5 1.79, m
1.91, mc
38.6
1.91, mc
4.13, mc
69.5 4.12, mc
43.4 1.95, mc
41.4 2.13, td (8.5, 4.6)
69.5
43.6
41.4
1.95, mc
2.14, td (8.5, 4.5)
4.20, mc
63.2 4.20, dd (10.9, 7.5) 63.2
4.08, dd (10.9, 7.0)
10b
4.08, mc
11a
11b
4.22, d (11.1)
4.00, d (11.1)
68.0 4.15, d (11.1)
3.89, d (11.1)
67.5
isovaleryl
-COO-
-CH2-
-CH<
(-CH3)a
(-CH3)b
sugar
1′
171.0
171.0
42.3
24.8
20.8
Compound 6 was isolated as a white, amorphous solid. The
HRFABMS spectrum displayed a quasimolecular ion at m/z 447.22
2.20, m
42.3 2.20, m
24.8 1.98, mc
20.8 0.891, d (6.6)
0.890, d (6.6)
1.97, mc
1
([M + H]+), consistent with the chemical formula C21H34O10. H,
0.891, d (6.6)
0.890, d (6.6)
COSY, HSQC, HMBC, and ROESY NMR spectra showed that
the aglycon part of the molecule was identical to that of compound
4. The HSQC spectrum also exhibited one anomeric signal (δC
100.1, δH 4.90), revealing the presence of a sugar moiety at C-11
(δ 68.6) as determined by analysis of the HMBC spectrum.
Interestingly, the COSY spectrum showed correlations between the
anomeric proton (δ 4.90) and two protons (δa 2.56, δb 2.11) of a
methylene group, which was assigned to C′-2 of the sugar moiety.
Extensive analysis of the COSY spectrum enabled determination
of the connectivity of the sugar residue, which was consistent with
a 2-deoxypyranose. The 13C and 1H spectroscopic data were
comparable to those of 2-deoxy-ꢀ-D-glucopyranose (see Supporting
Information). The ꢀ glycosidic linkage was deduced from the
coupling constants of the anomeric proton (9.7, 1.8 Hz).24 Acid
hydrolysis followed by derivatization with L-cysteine methyl ester
hydrochloride and GC analysis proved the absolute configuration
of the sugar moiety to be 2-deoxy-D-glucose. Therefore, the
structure of 6 was elucidated as patrinoside-aglycone-11-O-2′-
deoxy-ꢀ-D-glucopyranoside.
4.53, d (8.0)
4.19, dd (8.0, 1.6)
100.8 4.27, d (7.9)
77.6 3.24, dd (8.9, 7.9)
204.6 3.34, mc
100.3
75.4
77.5
70.0
76.5
60.7
2′
3′
4′
4.10, mc
72.2 3.07, mc
5′
3.29, ddd (10.0, 5.2, 1.9) 76.2 3.10, mc
6′a
3.74, m
3.59, m
4.78, d (1.3)
3.83, m
3.66, m
4.77, t (9.8)
3.90, dq (9.8, 6.3)
0.99, d (6.3)
60.5 3.67, m
6′b
3.45, m
1′′
98.1 5.20, d (1.3)
69.6 3.72, m
67.7 3.62, m
73.4 4.75, t (9.8)
65.7 4.01, dq (9.9, 6.3)
17.2 0.96, d (6.3)
99.0
70.0
67.8
73.7
65.1
17.2
2′′
3′′
4′′
5′′
6′′
acetyl
10-COO-
10-CH3
4′′-COO-
4′′-CH3
170.4
170.3
20.6d
170.1
20.6d
1.99, sd
2.00, sd
20.6d 1.99, sd
169.9
20.6d 2.00, sd
a Recorded at 600 MHz. b Assignments based on 2D HSQC and
HMBC spectra recorded at 150 MHz. c Overlapping signals. d Signals in
the same column may be interchanged.
Compounds 1-4 represent the first examples of acylated iridoid
diglycosides bearing the D-ribohexo-3-ulopyranosyl sugar moiety.
The occurrence of D-ribohexo-3-ulose as a sugar moiety in
glycosides isolated from natural sources is relatively rare and has
been hitherto reported for five iridoid glycosides,15,25-27 one acyclic
monoterpene glycoside,28 one dihydrochalcone glycoside,16 three
cardenolide glycosides,17,29 and two indoxyl glycosides.18 To the
best of our knowledge, compound 6 is the first example of an iridoid
glycoside bearing the 2-deoxy-D-glucopyranosyl sugar moiety.
Compounds 1-6 were also identified in leaves of S. ebulus collected
in July 2009 by means of HPLC-MS (see Supporting Information).
649.27 ([M + H]+), consistent with the chemical formula C29H44O16.
Thus, compounds 4 and 3 were isomers. The 13C and 1H
spectroscopic data of the sugar moieties (Table 2) showed very
good correspondence with those of 1 and confirmed the presence
of the same disaccharide moiety, linked to C-11, as demonstrated
by the HMBC spectrum. Also, acid hydrolysis followed by
derivatization and GC analysis provided results identical to
compound 1. Concerning the aglycon part of the molecule, the
upfield values for CH2-10 (δC 62.0, δH 4.41, 4.33), compared to 1
(δC 64.5, δH 4.83, 4.68), suggested the absence of an acetyl group
at this position. This was confirmed by analysis of the HMBC
spectrum, which showed no correlations between H2-10 (δa 4.41,
δb 4.33) and quaternary carbons. 1H and ROESY NMR data were
in agreement with the relative configurations of C-1, C-5, C-7, C-8,
and C-9 of the aglycon part as assigned for compound 1. Therefore,
the structure of 4 was elucidated as patrinoside-aglycone-11-O-
[4′′-O-acetyl-R-L-rhamnopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopy-
ranoside].
Experimental Section
General Experimental Procedures. All reagents used were of
analytical grade and were purchased from Sigma Aldrich (Vienna,
Austria). HPLC solvents were of gradient grade. Technical-grade
solvents were distilled before use. Water was produced by reverse
osmosis followed by distillation. Optical rotations were measured using
a Perkin-Elmer (Wellesley, MA) 341 polarimeter. FTIR spectra were
recorded on a Bruker (Bruker Optics, Ettlingen, Germany) IFS 25 FTIR
spectrometer in transmission mode (4000-600 cm-1) using ZnSe disks
of 2 mm thickness. NMR spectra were acquired using Bruker (Bruker
Biospin, Rheinstetten, Germany) DRX 300 and Avance II 600
spectrometers using C5D5N or DMSO-d6 (containing 0.03% TMS)
(Euriso-Top, Saint-Aubin, France). Chemical shift values were refer-
enced to the residual solvent signals. HRFABMS spectra were recorded
in positive mode using a Finnigan (San Jose, CA) MAT 95-S
spectrometer (Cs-Gun, 20 keV, matrix: glycerine). LC analyses were
carried out using an HP 1100 system (Agilent, Waldbronn, Germany)
equipped with autosampler, DAD, and column thermostat. Separations
Compound 5 was isolated as a white, amorphous solid. The
HRFABMS spectrum displayed a quasimolecular ion at m/z 693.30
([M + H]+), consistent with the chemical formula C31H48O17 and
thus revealed one degree of unsaturation lower compared to 1.
Except for an additional oxymethine signal (δC 79.7, δH 4.24) and
the lack of a ketone resonance, 13C and 1H NMR signals as well as
ROESY correlations of compounds 1 and 5 were almost identical.
Accordingly, ꢀ-D-ribohexo-3-ulopyranose had been replaced by
1
another sugar moiety. Corresponding H and 13C signals of this

1802 Journal of Natural Products, 2009, Vol. 72, No. 10
Pieri et al.
were performed on a Phenomenex (Torrance, CA) Synergi Max-RP
80A column (150 × 4.60 mm i.d., 4 µm) and a Merck (VWR,
Darmstadt, Germany) LiChroCART 4-4 guard column with LiChro-
spher 100 RP18 (5 µm) packing. A mobile phase consisting of 0.025%
TFA in H2O (v/v) (solvent A) and a mixture MeCN-MeOH (1:1; v/v)
(solvent B) was employed with gradient elution (0 min, 70:30 (A:B);
50 min, 45:55; 55 min, 2:98; 60 min, 2:98). The detection wavelength
was 205 nm, and the thermostat was set at 35 °C. The injection volume
was 10 µL; the flow rate was 0.6 mL/min. For LC-ESIMS experiments,
the HPLC system was coupled to a Bruker (Bruker Daltonics, Bremen,
Germany) Esquire 3000plus iontrap, replacing solvent A with a solution
of 0.1% formic acid in H2O (v/v). The MS parameters were as follows:
split 1:5; ESI positive mode; spray voltage +4 kV; nebulizer gas 30
psi; drying gas flow rate 8.00 L/min; m/z range 100-1200. For HSCCC
separations a P.C. Inc. (Potomac, MD) series 690 multilayer (triple)
coil HSCCC instrument with a Gilson (Villiers-le-Bel, France) pump
system (model 302/803 C) was used. Semipreparative HPLC separations
were carried out on a Dionex (Dionex Softron, Germering, Germany)
system fitted with a P580 pump, a ASI-100 autosampler, a UVD 170U
detector, a Gilson 206 fraction collector, and a Waters (Milford, MA)
X-Terra Prep MS C18 column (100 × 7.8 mm i.d., 5 µm) or
Phenomenex AQUA 125A column (250 × 10.0 mm i.d., 5 µm). GC
analyses were carried out on a Perkin-Elmer autosystem GC fitted with
a FID detector. A PERMABOND SE-54 fused silica capillary column
(Macherey-Nagel, Du¨ren, Germany) (50 m × 0.32 mm i.d., 0.25 µm
film thickness) was used with helium as carrier gas (1 mL/min). The
injector and detector temperatures were kept at 280 and 300 °C,
respectively. The oven temperature program was set as follows: injection
at 65 °C; isothermal hold for 2 min; temperature increase of 6 °C/min
to 300 °C; isothermal hold for 15 min. The injection volume was 2.5
µL. Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden) and silica
gel (VWR, Darmstadt, Germany) were used as stationary phases for
CC. TLC was carried out on silica gel 60 F254 plates (VWR, Darmstadt,
Germany) using CHCl3-MeOH-H2O (100:30:3; v/v/v) as mobile
phase, and detection was performed with vanillin/H2SO4 (1% w/v and
5% v/v methanolic solutions, respectively).
subjected to CC over Sephadex LH-20 (90 × 2 cm) using acetone as
mobile phase, affording 15 and 5 subfractions, respectively. Subfraction
10 derived from fraction D contained compound 4 (9.2 mg). Further
amounts (17.6 mg) of compound 4 were obtained from subfraction 2
derived from fraction E by semipreparative HPLC (X-Terra column;
isocratic elution, H2O-MeCN (75:25; v/v); flow rate 3 mL/min; column
temperature 35 °C). Fraction F (57.8 mg) was subjected to HSCCC
separation using EtOAc-2-propanol-H2O (1:0.2:1; v/v/v) as solvent
system in “tail-to-head” mode with the upper layer as mobile phase
(coil volume 325 mL; coil rotation 800 rpm; flow rate 1 mL/min) to
yield 26 subfractions. Compound 6 (6.9 mg) was found in subfractions
18-20.
10-O-Acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-r-L-rham-
nopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (1): white, amor-
phous solid; [R]20 -103.9 (c 0.31, MeOH); HPLC-online UV λmax
D
205; FTIR νmax (cm-1) 3423, 2960, 2934, 2875, 1739, 1668, 1600, 1575;
1H NMR (C5D5N and DMSO-d6, 600 MHz) see Tables 1 and 3; 13C
NMR (C5D5N and DMSO-d6, 75 and 150 MHz) see Tables 1 and 3;
ESIMS m/z 709 [M + H2O + H]+; HRFABMS m/z 691.28 [M + H]+
(calcd for C31H47O17, 691.28).
7-O-Acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-r-L-rham-
nopyranosyl-(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (2): white, amor-
phous solid; [R]20D -86.1 (c 0.30, MeOH); HPLC-online UV λmax 205;
FTIR νmax (cm-1) 3382, 2961, 2935, 2875, 1738, 1668, 1602, 1573;
1H NMR (C5D5N and DMSO-d6, 600 MHz) see Tables 1 and 4
(Supporting Information); 13C NMR (C5D5N, 75 MHz) see Table 1;
ESIMS m/z 709 [M + H2O + H]+; HRFABMS m/z 691.28 [M + H]+
(calcd for C31H47O17, 691.28).
10-O-Acetylpatrinoside-aglycone-11-O-[r-L-rhamnopyranosyl-
(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (3): white, amorphous solid;
[R]20 -67.8 (c 0.29, MeOH); HPLC-online UV λmax 205; FTIR νmax
D
1
(cm-1) 3381, 2960, 2932, 2875, 1738, 1667, 1600, 1575; H NMR
(C5D5N, 600 MHz) see Table 1; 13C NMR (C5D5N 75 MHz) see Table
1; ESIMS m/z 667 [M + H2O + H]+; HRFABMS m/z 649.27 [M +
H]+ (calcd for C29H45O16, 649.27).
Patrinoside-aglycone-11-O-[4′′-O-acetyl-r-L-rhamnopyranosyl-
(1f2)-ꢀ-D-ribohexo-3-ulopyranoside] (4): light yellow, amorphous
solid; [R]20D -111.1 (c 0.32, MeOH); HPLC-online UV λmax 205; FTIR
Plant Material. Leaves of Sambucus ebulus were collected near
Magdalensberg, Carinthia, Austria, in September 2003. A voucher
specimen (CS-09200301) was deposited at the Herbarium of the Institut
fu¨r Pharmazie/Pharmakognosie, Leopold-Franzens-Universita¨t Inns-
bruck, Austria.
ν
max (cm-1): 3374, 2960, 2933, 2875, 1739, 1668, 1600, 1575; 1H NMR
(C5D5N and DMSO-d6, 600 MHz) see Tables 2 and 4 (Supporting
Information); 13C NMR (C5D5N, 75 MHz) see Table 2; ESIMS m/z
671 [M + Na]+; HRFABMS m/z 649.27 [M + H]+ (calcd for
C29H45O16, 649.27).
Extraction and Isolation. Air-dried leaves of S. ebulus (401.5 g)
were ground and extracted with 1.5 L of 96% EtOH at room temperature
(8 times). After removal of the solvent under reduced pressure, the
resulting crude extract (104.1 g) was suspended in 1 L of H2O and
successively extracted with petroleum ether (1 L × 8), diethyl ether (1
L × 8), EtOAc (1 L × 5), and n-BuOH (1 L × 5). The resulting extracts
were concentrated under reduced pressure, affording petroleum ether
(27.60 g), diethyl ether (4.69 g), EtOAc (8.90 g), n-BuOH (21.77 g),
and H2O (44.33 g) extracts. The EtOAc extract (8.53 g) was subjected
to CC over Sephadex LH-20 (90 × 3.5 cm) eluting with MeOH to
yield nine fractions. Fraction 3 (3.045 g) was divided into three equal
parts of approximately 1 g each and separately subjected to HSCCC
using petroleum ether-EtOAc-96% EtOH-H2O (1:2:1:1; v/v/v/v) as
solvent system in “tail-to-head” mode with the upper layer as mobile
phase (coil volume 325 mL; coil rotation 800 rpm; flow rate 1 mL/
min). Thirty, 27, and 22 fractions were obtained, respectively. Fractions
containing compounds 1-6 were combined according to TLC and
HPLC chromatograms to yield six enriched fractions (A-F). Fraction
A (218.9 mg) was subjected to CC over silica gel (90 × 2 cm), affording
seven subfractions. Subfractions 4 (23.4 mg), 5 (65.2 mg), and 7 (27.0
mg) were separately fractionated by semipreparative HPLC (X-Terra
column; isocratic elution, H2O-MeCN (73:27; v/v); flow rate 3 mL/
min; column temperature 35 °C) to yield compound 1 (7.3, 12.8, and
12.4 mg, respectively). Fraction B (113.57 mg) was chromatographed
by CC over Sephadex LH-20 (90 × 2 cm) using acetone as mobile
phase, affording 11 subfractions. Compound 2 (11.4 mg) was purified
from subfraction 2 (34.9 mg) by semipreparative HPLC (X-Terra
column; isocratic elution, H2O-MeCN (75:25; v/v); flow rate 3 mL/
min; column temperature 35 °C). Fraction C (104.4 mg) was subjected
to CC over Sephadex LH-20 (90 × 2 cm), affording 11 subfractions.
Subfraction 4 (42.6 mg) was subjected to semipreparative HPLC
(AQUA column; isocratic elution, H2O-MeCN (70:30; v/v); flow rate
3 mL/min; column temperature 20 °C) to yield compounds 3 (5.0 mg)
and 5 (8.8 mg). Fractions D (82.1 mg) and E (95.0 mg) were separately
10-O-Acetylpatrinoside-aglycone-11-O-[4′′-O-acetyl-r-L-rham-
nopyranosyl-(1f2)-ꢀ-D-glucopyranoside] (5): white, amorphous solid;
[R]20 -70.2 (c 0.24, MeOH); HPLC-online UV λmax 205; FTIR νmax
D
1
(cm-1) 3395, 2960, 2933, 2875, 1739, 1667, 1580; H NMR (C5D5N
and DMSO-d6, 600 MHz) see Tables 2 and 3; 13C NMR (C5D5N and
DMSO-d6, 75 and 150 MHz) see Tables 2 and 3; ESIMS m/z 711 [M
+ H2O + H]+; HRFABMS m/z 693.30 [M + H]+ (calcd for C31H49O17,
693.30).
Patrinoside-aglycone-11-O-2′-deoxy-ꢀ-D-glucopyranoside (6): white,
amorphous solid; [R]20D -63.3 (c 0.28, MeOH); HPLC-online UV λmax
205; FTIR νmax (cm-1) 3374, 2958, 2929, 2873, 1746, 1666, 1593; 1H
NMR (C5D5N, 600 MHz) see Table 2; 13C NMR (C5D5N, 75 MHz)
see Table 2; ESIMS m/z 469 [M + Na]+; HRFABMS m/z 447.22 ([M
+ H]+ (calcd for C21H35O10, 447.22) and m/z 469.20 [M + Na]+ (calcd
for C21H34NaO10, 469.20).
Acid Hydrolysis of 1-6. Compounds 1-6 (1 mg each) were
subjected to hydrolysis with 1.5 mL of 1 N HCl (90 °C, 3 h). After
cooling, the reaction mixture was neutralized with NaOH and partitioned
three times with CHCl3. The H2O phase was then evaporated to dryness
and reconstituted in 2 mL of H2O.
Determination of L-Rhamnose, D-Glucose, and 2-Deoxy-D-glucose
(1-6). Solutions (1 mL each) derived from acid hydrolysis of 1-6
were evaporated to dryness and the residues extracted with 300 µL of
pyridine. L-Cysteine methyl ester hydrochloride (1 mg) was added, and
the resulting mixture was stirred at 60 °C for 1 h.21 Then, 150 µL of
a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)-trim-
ethylchlorosilane (TMCS) (99:1) was added, and the solution was stirred
at 60 °C for 30 min. After centrifugation, 2.5 µL of the supernatant
was subjected to GC analysis. Standards of L-rhamnose (0.2 mg),
D-glucose (0.2 mg), and 2-deoxy-D-glucose (0.2 mg) were derivatized
using the same procedure, showing chromatographic peaks at 36.44,

Iridoid Glycosides from Sambucus ebulus
Journal of Natural Products, 2009, Vol. 72, No. 10 1803
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analytical samples were co-injected with the derivatized standards,
showing matching peaks corresponding to L-rhamnose for compounds
1-4, L-rhamnose and D-glucose for compound 5, and 2-deoxy-D-glucose
for compound 6.
Determination of D-Ribohexo-3-ulose. To 1 mL of the solution
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30
derived from acid hydrolysis of 1-4, 1 mg of NaBH4 was added
while stirring. After 1 h, the reagent excess was eliminated by dropwise
addition of concentrated CH3COOH. The mixture was then concentrated
under reduced pressure. The excess boric acid was eliminated by
repeated addition of 10% CH3COOH in MeOH followed by concentra-
tion under reduced pressure (five times).31,32 The residue was then
extracted with 300 µL of pyridine, to which 150 µL of a mixture
BSTFA-TMCS (99:1) was added under stirring (60 °C, 30 min). After
centrifugation, 2.5 µL of the supernatant was subjected to GC analysis.
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using the same procedure, showing chromatographic peaks at 30.42
min (D-allitol) and 30.56 min (D-sorbitol), respectively. For identification
purposes, the analytical samples for compounds 1-4 were co-injected
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Acknowledgment. We thank Dr. J. M. Rollinger (Institute of
Pharmacy/Pharmacognosy, University of Innsbruck) for measuring the
FT-IR spectra, Dr. T. Mu¨ller (Institute of Organic Chemistry, University
of Innsbruck) for acquiring the HRFABMS spectra, and Dr. C. Seger
(Institute of Pharmacy/Pharmacognosy, University of Innsbruck) for
collecting the plant material.
Supporting Information Available: 1D and 2D NMR spectra of
compounds 1-6, 1H NMR data for the D-ribohexo-3-ulopyranosyl sugar
moiety in compounds 2 and 4 (DMSO-d6, 600 MHz), 1H and 13C NMR
data for 2-deoxy-ꢀ-D-glucose (C5D5N, 600 MHz), HPLC-MS com-
parison with plant material collected in July 2009, GC chromatograms
of the analysis of the sugar residues. This material is available free of
charge via the Internet at http://pubs.acs.org.
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