Flavonol glycosides from Chenopodium foliosum Asch

Article abstract of DOI:10.1016/j.phytol.2011.08.002

Three new flavonol glycosides, namely 6-methoxykaempferol-3-O-β- gentiobioside, gomphrenol-3-O-β-gentiobioside and gomphrenol-3-O-α-l- rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 6)]-β-d-glucopyranoside as well as the known patuletin-3-O-β- gentiobioside and spinacetin-3-O-β-gentiobioside were isolated from the aerial parts of Chenopodium foliosum Asch. The structures of the compounds were determined by means of spectroscopic methods (1D and 2D NMR, UV, IR, and HRMS). DPPH free radical scavenging activity of the new compounds was low or lacking.

Full text of DOI:10.1016/j.phytol.2011.08.002

Phytochemistry Letters 4 (2011) 367–371
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Flavonol glycosides from Chenopodium foliosum Asch
a
b
a
b
Zlatina Kokanova-Nedialkova , Daniel B u¨ cherl , Stefan Nikolov , J o¨ rg Heilmann ,
Paraskev T. Nedialkov *
a,
a
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Sofia, Dunav str. 2, 1000 Sofia, Bulgaria
b
Pharmaceutical Biology, Institute of Pharmacy, University of Regensburg, 93053 Regensburg, Germany
A R T I C L E I N F O
A B S T R A C T
Article history:
Three new flavonol glycosides, namely 6-methoxykaempferol-3-O-
b
-gentiobioside, gomphrenol-3-O-
-glucopyranosyl-(1 ! 6)]-
-gentiobioside and spinacetin-3-O-
b
-
Received 14 March 2011
Received in revised form 13 July 2011
Accepted 3 August 2011
Available online 22 August 2011
gentiobioside and gomphrenol-3-O-
a
-
L
-rhamnopyranosyl-(1 ! 2)[
b-D
b-D
-
glucopyranoside as well as the known patuletin-3-O-
b
b
-gentiobioside
were isolated from the aerial parts of Chenopodium foliosum Asch. The structures of the compounds were
determined by means of spectroscopic methods (1D and 2D NMR, UV, IR, and HRMS). DPPH free radical
scavenging activity of the new compounds was low or lacking.
Keywords:
ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Chenopodium
Amaranthaceae
6
-Methoxykaempferol
Gomphrenol
1
. Introduction
together with two known flavonol glycosides. The evaluation of
DPPH free radical scavenging capabilities of the new compounds
The genus Chenopodium L. (Amaranthaceae) comprises about
has also been done.
1
50 species, most of these are cosmopolites and are distributed
mainly in subtropical and temperate regions (Uotila and Tan,
997). Chenopodium is represented by nearly 18 species in the
2. Results and discussion
1
Bulgarian flora, mostly ruderal plants and widely distributed in the
country (Grozeva, 2007). Phytochemical investigations on Cheno-
podium species revealed the presence of triterpene saponins,
ecdysteroids, flavonoids and essential oils (Kokanova-Nedialkova
et al., 2009). Many species of this genus have been used
traditionally in indigenous systems of medicine for treatment of
numerous ailments. Biological properties of chenopods include
antimicrobial, cytogenetic, anthelmintic, cytotoxic, immunomod-
ulatory, hypotensive, haemagglutinative, trypanocidal and spas-
molytic activity (Yadav et al., 2007). Chenopodium foliosum Asch
has also been known in Bulgarian folk medicine as ‘‘garliche’’ or
A phytochemical investigation of C. foliosum led to the isolation
and structural identification of five flavonol glycosides 1–5. The
known compounds 1 and 5 were identified as patuletin-3-O-
gentiobioside and spinacetin-3-O- -gentiobioside (Fig. 1), respec-
tively by comparing their spectroscopic data with those reported in
b
-
b
1
13
the literature (Aritomi et al., 1985). The signals in the H and
C
spectra of the isolated compounds 2–4 were unambiguously
assigned using 2D NMR techniques, i.e., COSY, HSQC and HMBC.
1
Multiplicities were determined using H and HSQC spectra.
Determination of the sugars as
D-glucose and L-rhamnose was
done according the method of Noe and Freissmuth (1995) using
capillary electrophoresis.
‘‘svinski yagodi’’ (swine’s berries). The decoction of its aerial parts
has been used for treatment of cancer and as an immunostimulant.
Although some monoterpenes have been detected in the essential
oil from C. foliosum (Dembitsky et al., 2008) no detailed
phytochemical investigation of this plant has been undertaken
so far. The present study reports the isolation and chemical
characterization of two new flavonol gentiobiosides and a trioside
Compound 2 was isolated as optically active pale-yellow
crystalline powder. Its molecular formula was established as
+
C
6
28
H
32
O
17 by means of HRLSI–MS showing a [M + H] at m/z
41.1738. The IR spectrum showed absorption bands for hydroxyl
ꢀ
1
ꢀ1
groups (3458–3210 cm ), unsaturated carbonyl (1615 cm ) and
ꢀ
1
conjugated double bonds (1563, 1471 cm ). The UV spectrum
MeOH) of 2 was typical for 3-OH substituted flavonols. The
bathochromic shift of the maximum at 342 nm after addition of
AlCl /HCl ( = 22 nm) and NaOAc ( = 40 nm) indicated the
(
3
D
D
*
Corresponding author. Tel.: +359 2 9236 529; fax: +359 2 9879 874.
E-mail addresses: pnedialkov@pharmfac.net, pnedialkov@gmail.com
presence of free hydroxyl groups on 5 and 7, respectively
(Markham, 1982). A TLC analysis of sugar portion of compound
(
P.T. Nedialkov).
1
874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2011.08.002

3
68
Z. Kokanova-Nedialkova et al. / Phytochemistry Letters 4 (2011) 367–371
Fig. 1. Structures of the isolated compounds.
1
2
hydrolysate established the presence of glucose. The H NMR
Table 1
6
NMR data of compound 2 ( H 600 MHz; C 150 MHz; DMSO-d , 298 K).
1
13
a
spectrum (Table 1) was typical for a trifold-substituted A-ring
showing only a singlet at
due to OH-5 proton involved into a hydrogen bond. Two AB-type
H H
d 6.46. A broad singlet at d 12.64 was
Position
c
d , Mult.
d
H
(J in Hz)
HMBC
2
3
4
5
6
7
8
9
156.4, qC
132.8, qC
177.4, qC
152.2, qC
131.4, qC
coupling proton signals at
d
H
6.87 and 8.00, showed that position
0
4
3
of the B-ring was oxygenated. The three-proton singlet at
.73 gave cross-peak with C-6 (
d
H
d
C
131.4) in the HMBC experiment
12.64, br. s, OH
6.46, s
C6, C10
and was attributed to the methoxyl group at position 6. In
addition, two doublets at 5.35 (J = 7.6 Hz) and 4.02 (J = 7.7 Hz)
belonging to the anomeric glucosyl protons of configuration
appeared in the H NMR spectrum, as well. The former signal gave
cross-peak in the HMBC experiment with C-3 ( 132.8). This
b
158.3 , qC
d
H
94.1, CH
C4, C6, C7, C9, C10
b
151.8, qC
104.0, qC
121.0, qC
130.9, CH
115.1, CH
159.9, qC
1
1
1
2
3
4
6
1
2
3
4
5
6
0
0
d
C
0
0
0
0
0
0
and 6
and 5
8.00, d (8.9)
6.87, d (8.9)
C2, C4
evidence confirmed that sugar moiety was attached at position 3.
The signals of remaining sugar protons appeared mostly as double
doublets or triplets in the range of
0
0
C1 , C4
H
d 3.85–2.81. The coupling
-OCH
00
00
0
0
0
3
59.9, CH
3
3.73, s
C6
C-3, C-300
constants (J) ranging from 8.8 to 9.6 Hz were typical for axial/axial
101.1, CH
74.1, CH
76.2, CH
69.7, CH
76.3, CH
5.35, d (7.6)
1
3
00
00
00
00
coupling of glucose protons. The C NMR data of 2 (Table 1)
showed signals of a methoxy group, five hydrogen bearing
aromatic carbons as well as ten quaternary carbons that were
typical for 3-O-glycosidated 6-methoxykaempferols (Agrawal,
3.17, dd (9.0, 7.6)
3.22, t (9.0)
C1 , C3
0
0
0
00
C2 , C4
00
00
3.13, dd (9.6, 9.0)
3.29, ddd (9.6, 5.8, 1.5)
3.85, d (11.8), 3.44,
dd (11.8, 5.8)
4.02, d (7.7)
C3 , C5 , C6
00
C4
00
00 00
000
68.0, CH
2
C4 , C5 , C1
1
989). The gentiobiose-type linkage of the sugar moiety was
0
0
0
000
000
000
00
00
00
00
000
000
1
2
3
4
5
6
103.1, CH
73.4, CH
C6 , C2 , C3
confirmed by HMBC showing cross peaks between methylene
000
000
000
000
0
0
2.82,m
C1 , C3
protons (H-6 ) of inner glucose (
d
H
3.85 and 3.44) and anomeric
carbon (C-1 ) of terminal sugar ( 103.1). In addition, the
anomeric proton (H-1 ) of terminal glucose ( 4.02) correlated
with methylene carbon (C-6 ) of inner sugar ( 68.0). Thus,
compound 2 was identified as 6-methoxy-3,5,7,4 -tetrahydroxy-
flavon 3-O- -glucopyranoside or
-glucopyranosyl(1 ! 6)-O-
-methoxykaempferol-3-O- -gentiobioside (Fig. 1).
Compound 3 was isolated as optically active pale-yellow
crystalline powder. Its molecular formula was established as
c
000
76.6 , CH
2.93, t (8.8)
C2 , C4
0
00
d
C
000
000
69.7, CH
2.96, dd (9.2, 8.8)
2.80, m
C3 , C5 , C6
0
00
c
d
H
76.5 , CH
60.8, CH
–
000
C5
0
0
d
0
C
2
3.51, dd (11.7, 1.8), 3.34,
dd (11.7, 5.7)
a
b
-
D
b
-
D
Chemical shifts were referenced to the solvent signals (residual DMSO-d
39.51 for carbons).
6 H
at d
2
.50 for protons and
b
d
C
6
b-D
13
The signal was missing in C NMR spectrum, but it gave cross peak in HMBC
experiment.
c
The signals could not be assigned unambiguously to the certain carbon and may
be exchanged.
+
28 30
C H O17 by means of HRLSI–MS exhibiting a [M + H] at m/z

Z. Kokanova-Nedialkova et al. / Phytochemistry Letters 4 (2011) 367–371
369
6
39.1558. The IR spectrum showed absorption bands for hydroxyl
Similarly, the HMBC spectrum confirmed gentiobiose-type
linkage where the anomeric proton and carbon ( 102.9) of
the terminal glucose showed cross-correlation with methylene
ꢀ1
ꢀ1
groups (3361–3211 cm ), unsaturated carbonyl (1680 cm )
d
C
ꢀ
1
and conjugated double bonds (1561, 1480 cm ). The UV
spectrum of 3 in MeOH was typical for 3-OH substituted flavonols.
The bathochromic shifts of the maximum at 342 nm after addition
carbon (
d
C
67.7) and protons (
d
H
3.84 and 3.44) of the inner sugar,
0
respectively. Thus, 3 was identified as 6,7-methylenedioxy-3,5,4 -
of AlCl
3
/HCl (
D
= 30 nm) indicated the presence of free hydroxyl
trihydroxyflavone 3-O-
copyranoside or gomphrenol-3-O-b-D
Compound 4 was isolated as optically active pale-yellow
crystalline powder. Its molecular formula was established as
b
-
D
-glucopyranosyl-(1 ! 6)-O-
b-D-glu-
-gentiobioside (Fig. 1).
group at 5 position. On the other hand, the spectrum after addition
of NaOAc was almost identical to the one in MeOH which was
indication for blocked or missing 7-OH group (Markham, 1982). A
TLC analysis of sugar portion of compound 3 hydrolysate
+
34 40
C H O21 by means of HRLSI–MS showing a [M + H] at m/z
1
established the presence of glucose. The H NMR spectrum in
785.2140. The IR spectrum showed absorption bands for hydroxyl
ꢀ ꢀ1
1
DMSO-d
substituted ring B characterised by two doublets (J = 8.9 Hz,
J = 8.8 Hz), each integrating for two protons, at 8.03 and
12.58 ppm belongs to the
-OH group, involved in a hydrogen bond with the C-4 keto group
177.9, Table 2). A trisubstituted ring A carrying a methyle-
nedioxy group was indicated by a singlet signal at 6.85 for the
single aromatic proton and two doublets at 6.160 and 6.155
J = 1.0 Hz) for the methylenedioxy protons. In the HSQC spectrum
the latter protons showed a cross-peak with the carbon signal at
02.7 (Table 2). The HMBC experiment revealed a correlation
between methylenedioxy protons and the carbons at 6 ( 129.3)
153.9) position. The H and C NMR data of 3 were in
6
(Table 2) showed a typical flavonoid pattern with a para-
groups (3384 cm ), unsaturated carbonyl (1637 cm ) and conju-
ꢀ1
gated double bonds (1561, 1519 cm ). The sugar portion of
compound4 hydrolyzateshowed a majorspotonTLC corresponding
toglucoseaswellasone minorspotduetorhamnose. The UVspectra
d
H
d
H
6
5
.89 ppm. A broad singlet centred at d
H
1
13
and aglycone signals in H and C NMR spectra (Table 2) of 4 were
similar to those of compound 3 pointing to the same aglycone. The
(d
C
1
13
d
H
H, C and 2D NMR confirmed again the presence of a gentiobiosyl-
substituent at OH-3 (a cross-peak between 5.50 and 132.8 in
HMBC spectrum) substituted with a rhamnopyranosyl moiety. An
d
H
d
H
d
C
(
1
d
C
additional anomeric proton signal at
spectrum gave a HMBC correlation with C-2
glucopyranosyl moiety. The cross-peak between proton H-2
3.45) of primary glucose and anomeric carbon C-1
H
d 5.09 (J = 1.3 Hz) in H NMR
0
0
1
C
(d 77.0) of primary
0
0
d
C
(d
H
1
13
0000
and 7 (
d
C
C
(d 100.6) of
good agreement with literature data for 3-O-glycosidated 6,7-
rhamnopyranose was also observed. Accordingly, the sugar signals
0
13
methylenedioxy-3,5,4 -trihydroxyflavones (Kohda et al., 1990;
in C NMR were typical for triglycosides with primary
b
-
D
-
1
00
Mosquera et al., 2008). Additionally, in the H NMR spectrum of 3
glucopyranose glycosidated at C-2 (downfield shifted to
d
C
77.0)
-rhamnopyranose and
-glucopyranose (Kikuchi and Matsuda, 1996; Veitch et al., 2010).
00
appeared two doublets at
J = 7.7 Hz) indicating two sugar units. The downfield doublet
exhibits a cross-peak with the signal of C-3 ( 133.2) showing
d
H
5.36 (J = 7.2 Hz) and
d
H
3.99
and C-6 (downfield shifted to d 67.7) with a
C
(
b
d
C
Thus, the structure of 4 was established as 6,7-methylenedioxy-
1
3
0
clearly the position of glycosylation. The C NMR signal pattern of
3,5,4 -trihydroxyflavone 3-O-
a
-
L
-rhamnopyranosyl-(1 ! 2)[
b-D-
the sugar carbons (Table 2) was similar to that of compound 2.
glucopyranosyl-(1 ! 6)]-
b-D-glucopyranoside (Fig. 1).
Table 2
1
13
a
6
NMR data of compounds 3 and 4 ( H 600 MHz; C 150 MHz; DMSO-d , 298 K).
Position
3
4
d
c
, Mult.
d
H
(J in Hz)
HMBC
d
c
, Mult.
d
H
(J in Hz)
HMBC
2
3
4
5
6
7
8
9
156.9, qC
133.2, qC
177.9, qC
140.4, qC
129.3, qC
153.9, qC
89.6, CH
151.8, qC
107.1, qC
120.7, qC
130.9, CH
115.1, CH
160.0, qC
102.7, CH
100.8, CH
74.1, CH
76.2, CH
69.8, CH
76.6, CH
156.9, qC
132.8, qC
177.8, qC
140.5, qC
129.3, qC
153.8, qC
89.6, CH
151.8, qC
107.1, qC
120.7, qC
130.8, CH
115.1, CH
160.0, qC
102.7, CH
98.3, CH
77.2, CH
77.1, CH
70.2, CH
76.6, CH
12.58 br. s, OH
6.85, s
12.61, br. s.
6.83, s
C6, C7, C9, C10
C6, C7, C9, C10
1
1
2
3
4
0
0
0
0
0
0
0
0
0
0
0
0
0
and 6
and 5
8.03, d (8.9)
C2, C3 , C4 , C5
8.02, d (8.9)
C2, C3 , C4 , C5
0
0
0
0
6.89, d (8.9)
C1 , C4
6.87, d (8.9)
C1 , C4
10.19, br. s, OH
10.20, br. s., OH
6.16, d (0.8); 6.15, d (0.8)
5.50, d (7.7)
O–CH
2
–O
2
6.160, d (1.0); 6.155, d (1.0)
5.36, d (7.4)
C6, C7
2
C6, C7
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
00
1
2
3
4
5
6
1
2
3
4
5
6
1
2
3
4
5
6
C3,
C3, C3
00
00
00
00
0000
00
3.19, dd (9.0, 7.4)
3.21, dd (9.0, 8.5)
3.09, dd (9.6, 8.5)
3.29, ddd (9.6, 6.3, 1.5)
C1 , C5
3.45, dd (9.2, 7.7)
3.37, m
C1 , C1
00
00
00
00
C2 , C4
C1 , C2 , C4 , C6
00
00
00
C3
3.07, dd (9.3, 9.0)
3.30, m
C3
00
C4
C4
00
00
000
000
000
000
000
000
00
00
000
000
00
67.7, CH
2
3.84, dd (12.0, 1.5); 3.44, dd (12.0, 6.3)
3.99, d (7.7)
C4 , C5 , C1
67.7, CH
2
3.8, m; 3.38, m
3.95, d (7.7)
C1 , C4 , C5
00
00
00
00
00
00
000
000
000
000
000
000
00
000
000
102.9, CH
73.3, CH
76.4, CH
69.6, CH
76.5, CH
C6 , C3 or C5
102.9, CH
73.3, CH
76.5, CH
69.7, CH
76.4, CH
C-3 , C6
000
000
2.78, dd (9.0, 7.7)
C1 , C3
2.74, td (8.8, 7.7)
2.83, dd (9.0, 8.8)
2.94, dd (9.3, 9.0)
2.67, ddd (9.3, 5.5, 2.3)
3.46, m; 3.32, m
5.09, d (1.3)
C1 , C3
000
000
2.84, dd (9.0, 8.8)
C2 , C4
C2 , C4
000
000
000
2.96, dd (9.5, 8.8)
C3 , C6
C3 , C6
000
000
2.71, ddd (9.5, 5.5, 2.1)
3.47, dd (12.0, 1.7,); 3.33, dd (12.0, 5.5)
C4
C4
000
000
60.7, CH
2
C5 , C4
60.7, CH
2
00
0000
0000
0000
100.6, CH
70.6, CH
70.5, CH
71.8, CH
68.3, CH
C2 , C2 , C5
0000
3.74, dd (3.3, 1.3)
3.47, dd (9.5, 3.3)
3.14, t (9.5)
C3 , C4
0000
3.77, dd (9.5, 6.2)
0.82, d (6.2)
C4
0000
0000
17.4, CH
3
C5 , C4
a
Chemical shifts were referenced to the solvent signals (residual DMSO-d
6
at
d
H
2.50 for protons and d 39.51 for carbons).
C

3
70
Z. Kokanova-Nedialkova et al. / Phytochemistry Letters 4 (2011) 367–371
All isolated compounds share one common feature: an
3.3. Extraction and isolation
oxygenation at position 6, which was either methylated or
involved in methylenedioxy group. Although 6-methoxy flavo-
nols were common for some genera of Chenopodioideae
The aerial parts of C. foliosum were dried in the shade and
2 2
powdered plant material (857 g) was extracted with CH Cl
(
Sanderson et al., 1988) these compounds were rarely found in
(7 ꢁ 3 L). After filtration, the extracts were combined and the
solvent was evaporated under reduced pressure to give 31.3 g of
greenish waxy residue. Subsequently, the plant material was
extracted with MeOH (7 ꢁ 3 L), 70% aq. MeOH (6 ꢁ 2 L) and 50% aq.
MeOH (2 ꢁ 2 L). The resulting extracts were combined, concen-
trated under vacuo until most of the MeOH was removed and the
Chenopodium species (Kokanova-Nedialkova et al., 2009). Within
Amaranthaceae gomphrenol has been found only in species
belonging to subfamily Gomphrenoideae, so far. The occurrence of
6
-methoxykaempferol, spinacetin, patuletin and gomphrenol
derivatives in Chenopodium genus is reported here for the first
time. More detailed survey on distribution of 6-methoxy and 6,7-
methylenedioxy flavonols within Amaranthaceae is required in
order to establish the significance of these compounds as
chemotaxonomic markers.
aq. residue was successfully extracted with CH
The aq. layer was conc. to 200 mL and then subjected to CC over
O–MeOH
2
Cl
2
(8 ꢁ 300 mL).
Diaion HP-20 (7 cm ꢁ 75 cm) with eluent
H
2
(100:0 ! 0:100) to obtain 86 fractions (500 mL each) that were
combined into 23 pooled fractions (I–XXIII) on basis of the TLC
profiles. The fractions XIII (2.46 g, 60% MeOH) and XIV (7.37 g, 70%
MeOH) were separately subjected to CC over MCI gel
The new compounds 2, 3 and 4 were tested for DPPH free radical
scavenging activity at 100
were not active while compound 3 showed weak DPPH free radical
mM (Blois, 1958). Compounds 2 and 4
scavenging activity (18.0%) compared to vitamin C (97.4%) and BHT
(4 cm ꢁ 30 cm, 50 mL fraction volume) with eluent H
2
O–MeOH
(48.8%) at the same concentration.
(100:0 ! 0:100). The combined fractions 27–31 (0.70 g, 50%
MeOH) of XIV was chromatographed over RP-18 (4 cm ꢁ 25 cm,
3
. Experimental
50 mL fraction volume) with H
isocratic semi-prep. HPLC purification of sub-fractions 20–25
2
O–MeOH (100:0 ! 0:100). An
ꢀ1
3.1. General
(0.23 g, 40–50% MeOH) with MeOH–H
80 nm) as eluent gave pure 1 (8 mg) and 2 (11 mg). The fractions
2
O (38:62, 19.5 mL min
,
2
Melting points (m.p.) were measured on a Kofler hot-stage
32–35 (60% MeOH) of XIII and 32–34 (60% MeOH) of XIV were
combined together on the basis of TLC analysis. This combined
fraction (2.39 g) was further subjected to a CC over RP-18 and was
microscope and were uncorrected. Optical rotations (OR) were
measured on a Schmidt + Haensch UniPol L1000. Infrared (IR)
spectra were recorded on a Bruker Tensor 27 spectrophotometer
equipped with ATR accessory. UV spectra were run in MeOH or
with the standard shift reagents (Markham, 1982) on a Varian
Cary 50 spectrophotometer (Palo Alto, USA). ESI–MS spectra were
measured on a ThermoQuest Finnigan TSQ 7000 (4 kV). HRLSI–MS
eluted with H
fraction 23–26 (0.75 g, 40–50% MeOH) was further separated by
semi-prep. HPLC, isocratically eluted with MeOH–H O (40:60) and
2
O–MeOH (100:0 ! 0:100). The resulted sub-
2
yielded compounds 3 (37 mg), 4 (59 mg), 5 (15 mg).
+
0
spectra were recorded on a Finnigan MAT 95 (glycerin, Cs , 20 kV).
3.3.1. 6-Methoxy-3,5,7,4 -tetrahydroxyflavone 3-O-
b
-
D-
NMR spectra were recorded on a Bruker BioSpin (Rheinstetten,
Germany) Avance III 600 spectrometer at 600 MHz ( H) and
glucopyranosyl-(1 ! 6)-
b-D-glucopyranoside (2)
1
Pale-yellow crystalline powder from MeOH–H
2
O; m.p. 230–
max (MeOH) log ( ), 342
) 369, 307sh, 277, (+AlCl /HCl) 364,
307sh, 278, (+NaOAc) 382, 307sh, 274, (+NaOAc/H BO ) 356, 272;
1
3
21.9
1
50 MHz ( C) in DMSO-d
carried out with Diaion HP-20, MCI-gel (Supelco, USA) and
LiChroprep C-18 (40–63 m, using an over-pressure of 0.8–
.0 bar, Merck, Darmstadt, Germany) as stationary phase. Semi-
6
. Column chromatography (CC) was
231 8C; [
a
]
D
ꢀ138 (c = 0.1; DMSO); UV
l
e
(4.30), 270 (4.25), (+AlCl
3
3
m
3
3
ꢀ
1
1
IR
(C 55 C); H NMR (600 MHz, DMSO-d
(150 MHz, DMSO-d
nmax (ATR) cm 3458–3210 (OH), 1615 (C 55 O), 1563, 1471
1
13
preparative high performance liquid chromatography (HPLC) was
performed on a Waters (Milford MA, USA) Breeze 2 high pressure
binary gradient system consisting of a pump model 1525EF,
manual injector 7725i and an UV detector model 2489. Separa-
tions were achieved on a semi-preparative HPLC column Kromasil
6
) see Table 1; C NMR
+
6
) see Table 1; ESI–MS m/z 641 [M + H] ; HRLSI–
+
MS found m/z 641.1738 [M + H] ; calcd. for C28
641.1718.
H
33
O
17 m/z
0
C18 (250 mm ꢁ 21.6 mm, 10
m
m) purchased from Eka Chemicals
3.3.2. 6,7-Methylenedioxy-3,5,4 -trihydroxyflavone 3-O-
b
-D
-
AB (Bohus, Sweden). Determination of the absolute stereochem-
istry of the sugars was done on a Biofocus 3000 apparatus
glucopyranosyl-(1 ! 6)-
b-D-glucopyranoside (3)
Pale-yellow crystalline powder from MeOH–H
2
O; m.p. 235–
max (MeOH) log ( ), 342
) 378, 297, (+AlCl /HCl) 372, 297,
(+NaOAc) 339, 289, (+NaOAc/H BO ) 346, 278; IR max (ATR)
cm 3361–3211 (OH), 1680 (C 55 O), 1561, 1480 (C 55 C); H NMR
2
2.1
(
Biorad). Thin layer chromatography (TLC) was performed on
silica gel 60 F254 plates (Merck) using following mobile phases:
?–AcOH–MeOH–BuOH–CHCl (2:1:6:2:10), EtOAc–AcOH–
HCOOH–H (100:11:11:27) and EtOAc–AcOH–HCOOH–H
25:3:3:7). The chromatograms were observed under an UV light
254 and 366 nm) before and after spraying with 1% Natural
236 8C; [
a
]
D
ꢀ86 (c = 0.1; DMSO); UV
l
e
(4.27), 278 (4.10), (+AlCl
3
3
H
2
3
3
3
n
ꢀ
1
1
2
O
2
O
1
3
(
(
(600 MHz, DMSO-d
6 6
) see Table 2; C NMR (150 MHz, DMSO-d )
+
see Table 2; ESI–MS m/z 639 [M + H] ; HRLSI–MS found m/z
+
Product Reagent A (Carl Roth, Germany) in MeOH. All solvents
were of HPLC grade and were purchased from Merck or Sigma–
Aldrich (Taufkirchen, Germany). All reagents were of analytical
grades.
31
639.1558 [M + H] ; calcd. for C28H O17 m/z 639.1561.
0
3.3.3. 6,7-Methylenedioxy-3,5,4 -trihydroxyflavone 3-O-
a
b-D-
-L-
rhamnopyranosyl-(1 ! 2)[
b-
D-glucopyranosyl-(1 ! 6)]-
glucopyranoside (4)
3
.2. Plant material
Pale-yellow crystalline powder from MeOH–H O; m.p. 193–
2
2
2.3
1
94 8C; [
(4.29), 278 (4.12), (+AlCl
a
]
D
ꢀ210 (c = 0.1; DMSO); UV
l
max (MeOH) log ( ), 340
) 379, 298, (+AlCl /HCl) 373, 299,
(+NaOAc) 337, 284, (+NaOAc/H BO ) 342, 277; IR max (ATR)
3384 (OH), 1637 (C 55 O), 1560, 1519 (C 55 C); H NMR
e
Aerial parts of C. foliosum Asch were collected from Beglika,
3
3
Western Rhodopes, Bulgaria from June to September 2007, at an
altitude of 1600 m. The plant was identified and a voucher
specimen (No. SOM-Co-1207) was deposited at the National
Herbarium, Institute of Biodiversity and Ecosystems Research,
Bulgarian Academy of Sciences, Sofia, Bulgaria.
3
3
n
ꢀ
1
1
cm
(600 MHz, DMSO-d
see Table 2; ESI–MS m/z 785 [M + H] ; HRLSI–MS found m/z
1
3
6
6
) see Table 2; C NMR (150 MHz, DMSO-d )
+
+
41
785.2166 [M + H] ; calcd. for C34H O21 m/z 785.2140.

Z. Kokanova-Nedialkova et al. / Phytochemistry Letters 4 (2011) 367–371
371
3
3
.4. Sugar analysis
adsorption by the test samples and compared with that exercised
by vitamin C and BHT used as standards. All determinations were
performed in triplicate (n = 3).
.4.1. TLC analysis
2
Compounds 2–4 (2.0 mg) were dissolved in 2 N HCl (H O–
MeOH 1:1, 2 mL) and hydrolysed by heating at 100 8C for 2 h. After
evaporation of the solvent under vacuo, the residue was sonificated
Acknowledgement
in H
The water phase was evaporated under vacuum and redissolved in
0% MeOH. The sugar portions were co-chromatographed on a TLC
with authentic samples ( -glucose and -rhamnose). Mixtures
of EtOAc–pyridine–H O (12:5:4) on cellulose (twofold develop-
ment) and EtOAc–MeOH–H O–AcOH (13:3:3:4) on silica gel were
2
O (3 mL) and the mixture extracted with EtOAc (3 ꢁ 5 mL).
This study was supported by Grant 16-D/2008 from the Medical
Science Council at the Medical University of Sofia.
5
b
-D
a-L
2
Appendix A. Supplementary data
2
used as mobile phase. The spots were visualized by spraying with
anisidine-phthalate reagent (a solution of 1.23 g p-anisidine and
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytol.2011.08.002.
1
.66 g phthalic acid in 100 mL 95% EtOH) and heating the plate for
1
0 min (110–120 8C).
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3.4.2. CE determination of
L-rhamnose and D-glucose
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1
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the Schiff base, which was immediately reduced to the corre-
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derivatized compounds.
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L- sugar references (1 mg per sample) were
m
L of 0.1 M S-(ꢀ)-1-phenylethyamin to afford
m
m
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4
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3
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