Journal of Natural Products
Article
column, operated at 30 °C. Each 10 min chromatographic run was
carried out at a flow rate of 0.3 mL/min with a binary mobile phase
collected (C1−C6). Subfraction C3 was subjected to LPLC over an
ODS column (2.4 × 30 cm), eluting with MeOH−H O (32:68, v/v),
2
consisting of MeOH + 0.1% HCOOH (A) and 10 mM NH COOH +
followed by isocratic semipreparative HPLC, eluting with a mobile
4
0
.1% HCOOH (B) using a step gradient profile of 50% A for 0.5 min,
phase of MeCN−H O (10:90, v/v), to give compounds 1 (12 mg)
2
increasing up to 100% A in 5 min, held isocratic at 100% for 0.5 min,
then brought back down to 50% A over 0.1 min. After re-equilibration
at 50% A for 3.9 min, the next sample was injected.
TLC was carried out on precoated silica gel plates (Kieselgel G,
F254, 60, Merck, Darmstadt, Germany) with the solvent systems
EtOAc−HCOOH−H O (10:1:4), EtOAc−HCOOH−AcOH−H O
and 6 (11 mg). Subfraction C2 afforded compounds 2 (13 mg) and 3
(5.5 mg) using the same separation procedure as that used for fraction
C3. Fraction III was chromatographed over Sephadex LH-20 (4.7 × 50
cm), eluting with MeOH, to give six subfractions (A1−A6).
Subfraction A4 was purified by repeated LPLC over ODS C (2.4
18
2
2
× 30 cm), eluting with MeOH−H O (40:60, v/v), and was further
2
(
32:3:2:6), and EtOAc−EMK−HCOOH−H O (5:3:1:1). Spots
subjected to isocratic semipreparative HPLC, eluting with the mobile
2
were visualized under UV light (365 nm) by spraying with NTS/
PEG reagent. Column chromatography (CC) was performed using
Diaion HP-20 (Supelco, USA) and Sephadex LH-20 (Pharmacia Fine
Chemicals AB, Uppsala, Sweden). For low-pressure liquid chromatog-
raphy (LPLC), silica gel (MN Kieselgel 60, 0.04−0.063 mm,
phase MeCN−H O (14:86, v/v), to give compound 7 (45 mg).
2
Fraction IV was purified by CC over Sephadex LH-20, eluting with
MeOH, and four subfractions were collected (B1−B4). Further
separation of subfraction B2 was performed by repeated LPLC over an
ODS column (2.4 × 30 cm), eluting with MeOH−H O (35:65, v/v),
2
Macherey-Nagel, Du
̈
ren, Germany) and octadecylsilyl (ODS) gel
followed by isocratic semipreparative HPLC, eluting with the mobile
DAVISIL (Grace Davison Discovery Sciences, Hesperia, CA, USA)
were used. Semipreparative HPLC was performed on a Waters
phase MeCN−H O (14:86, v/v), which afforded compound 8 (48
2
mg). In a similar manner to that described for subfraction B2,
(
Milford, MA, USA) high-pressure binary gradient system consisting
of a pump model 1525EF, manual injector 7725i, UV detector model
489, and the software Breeze 2, using a Luna prepacked
semipreparative ODS column (100 Å, 250 × 10 mm, 5 μm,
Phenomenex, Torrance, CA, USA) and eluting with H O−o-H PO
subfraction B3 was fractionated to yield compounds 9 (17.4 mg) and
1
3 (5.1 mg).
2
The EtOAc extract (5.56 g) was chromatographed over a silica gel
column, eluting with a gradient of CH Cl −MeOH-H O (90:10:0 →
2
2
2
2
3
4
80:20:3, v/v/v), to obtain 13 fractions (Et1−Et13). Fraction Et4 was
0
.05%−MeCN at a constant flow rate of 4.5 mL/min. For HPLC
purified by repeated LPLC over an ODS column, eluting with a
analysis, a Luna prepacked ODS column (100 Å, 250 × 4.6 mm, 5 μm,
gradient of MeOH−H O (50:50 → 0:100, v/v), and five subfractions
2
Phenomenex) eluted with H O−o-H PO 0.05%−MeCN at a constant
flow rate of 1 mL/min was used. All chromatograms were monitored
at 254 and 330 nm.
GC-MS analysis of (2R)-2-butyl glycosides was performed using an
Agilent 7890A GC system interfaced to an Agilent 5975C MSD
operating at 70 eV, ion source temperature 230 °C, interface
temperature 280 °C. A split injection (1 μL injection volume, split
ratio, 20:1) at 270 °C injector temperature was utilized. A fused silica
capillary column, 5% phenyl/95% methyl polysiloxane (HP-5MS 30 m
2
3
4
were collected (Et4 −Et4 ). Subfraction Et4 was further subjected to
1
5
4
isocratic semipreparative HPLC, eluting with a mobile phase of
MeCN−H O (19:81, v/v), to obtain compounds 10 (4.5 mg), 11 (6.1
2
mg), and 12 (4.8 mg).
The air-dried plant material of A. monspessulanus ssp. illyricus (95 g)
was subjected to extraction with CH Cl to remove the lipophilic
2
2
constituents. The defatted plant material was exhaustively extracted
with a gradient of MeOH−H O (100:0 → 80:20, v/v) at room
2
temperature. The extract was filtered, and the solvent was removed
×
250 μm × 0.25 μm, Agilent J & W, USA), was used. The
under reduced pressure, yielding 19.6 g of solid residue. The residue
temperature program was as follows: 100 °C, then 270 °C at 3 °C/
min. The carrier gas was helium 5.6 at a flow rate of 1.4 mL/min. Data
acquisition was performed with Agilent GC/MSD ChemStation
version E.02.02 for the mass scan range 40−600 u.
The chemicals used in the pharmacology experiments were
pentobarbital sodium (Sanofi, France); NaCl, KCl, NaHCO , CaCl ·
was suspended in H O (100 mL) and fractionated by CC with Diaion
2
HP-20 (4.7 × 45 cm) to give six main fractions (A−F). Fraction C was
subjected to repeated LPLC over an ODS column (2.4 × 30 cm),
eluting with MeOH−H O (30:70, v/v), and four subfractions were
2
collected (C1−C4). Subfraction C2 was purified by CC over Sephadex
LH-20 (2.5 × 50 cm) followed by isocratic semipreparative HPLC,
3
2
2
H O, and D-glucose (Merck, Germany); KH PO (Scharlau Chemie
2
2
4
eluting with the mobile phase MeCN−H O (17:83, v/v), to afford
2
SA, Spain); MgSO ·7H O (Fluka AG, Germany); HEPES, collagenase
4
2
compounds 4 (29 mg) and 5 (6 mg).
from Clostridium histolyticum type IV, albumin, bovine serum fraction
V minimum 98%, EGTA, tert-butyl hydroperoxide (Sigma-Aldrich,
Germany).
Quercetin-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyra-
nosyl-(1→6)]-β-D-galactopyranosyl]-7-O-β-D-glucopyranoside (1):
orange, amorphous powder; UV (MeOH) λ (log ε) 250 (4.04),
max
Plant Material. The aerial parts of A. monspessulanus ssp.
monspessulanus were collected in May 2010 from Rodopi Mountain
close to the town of Dzhebel, Bulgaria, at coordinates 41°32′56.00″ N,
1
2
63 (sh) (3.87), 351 (3.74) nm; H NMR (pyridine-d , 400 MHz), see
Table 1; C NMR (pyridine-d , 100 MHz), see Table 1; HREIMS m/
z 963.2650 (calcd. for C H O , 963.2612 [M + HCOO] ).
5
13
5
−
40
51 27
2
5°21′47.43″ E, whereas the aerial parts of A. monspessulanus ssp.
N-(8-Methylquercetin-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-
illyricus were collected in May 2013 from a rocky area in the western
parts of Stara Planina Mountain close to the town of Vratsa, Bulgaria,
at coordinates 43°8′5.31″ N, 23°29′9.23″ E. The plants were identified
by Dr. D. Pavlova from the Department of Botany, Faculty of Biology,
Sofia University, where voucher specimens have been deposited (A.
monspessulanus ssp. monspessulanus, N SO 107533, and A.
monspessulanus ssp. illyricus, N SO 107532).
rhamnopyranosyl-(1→6)]-β-D-galactopyranosyl])-3-hydroxypiperi-
din-2-one (2): orange, amorphous powder; UV (MeOH) λ (log ε)
max
1
251 (4.13), 263 (sh) (3.97), 293 (sh) 3.60), 355 (3.96) nm; H NMR
13
(pyridine-d , 400 MHz), see Table 1; C NMR (pyridine-d , 100
5
5
MHz), see Table 1; HREIMS m/z 882.2695 (calcd for C H NO ,
3
9
48
22
−
882.2673 [M − H] ).
N-(8-Methylkaempferol-3-O-[α-L-rhamnopyranosyl-(1→2)-[α-L-
rhamnopyranosyl-(1→6)]-β-D-galactopyranosyl])-3-hydroxypiperi-
Extraction and Isolation. The air-dried powdered plant material
from A. monspessulanus ssp. monspessulanus (280 g) was exhaustively
extracted with 80% MeOH under reflux (23 × 750 mL). The extracts
were filtered and concentrated under reduced pressure. The
hydrophilic residue was dissolved in H O and extracted with
CH Cl to remove the lipophilic constituents. The defatted water
din-2-one (3): pale yellow, amorphous powder; UV (MeOH) λmax (log
1
ε) 259 (4.05), 293 (sh) (3.82), 345 (3.99) nm; H NMR (pyridine-d
,
5
400 MHz), see Table 1; 13C NMR (pyridine-d
, 100 MHz), see Table
48 NO21, 866.2724 [M −
5
1; HREIMS m/z 866.2747 (calcd. for C39
H] ).
H
2
−
2
2
residue was successively extracted with EtOAc and n-BuOH. The n-
BuOH extract was evaporated to dryness to give a solid residue (53.6
g). Part of the extract (50 g) was subjected to CC over Diaion HP-20
Determination of Absolute Configuration of Sugars. The
analysis was carried out by GC-MS according to the method by
Reznicek et al. including acidic hydrolysis and preparation of (2R)-2-
2
7
(
4.7 × 45 cm), eluting with H O−MeOH (100:0 → 0:100, v/v), to
butyl glycosides. Compounds 1−5 (2 mg) were hydrolyzed with
2
give nine main fractions (I−IX). Fraction II was chromatographed
concentrated AcOH (3.5 mL), concentrated HCl (1 mL), and H O
2
over Sephadex LH-20, eluting with MeOH, and six subfractions were
(5.5 mL) at 100 °C for 2 h. The mixture was extracted with EtOAc,
F
J. Nat. Prod. XXXX, XXX, XXX−XXX
Krasteva, Ilina
