5
58
Y.-M. Liu et al. / Phytochemistry Letters 6 (2013) 556–559
Fig. 2. Equilibrium between structure 1 and its deprotonated forms 1a or 1b.
compound 1 and 2 were very easy to be differentiated from other
constituents in N. glandulifera, so compound 1 and 2 would be
highly applicable as chemical markers of N. glandulifera in TLC or in
FLD-HPLC analysis.
we believe that these are promising candidates for further
investigation.
3. Experimental
Alkaloids with an indazole nucleus are rare in nature and
hitherto confined to only four analogs (Ali et al., 2008; Atta-ur-
Rahman et al., 1985, 1995; Liu et al., 2004). Thus, compounds 1 and
3.1. General experimental procedures
2
will be the fifth and sixth indazole-type alkaloids respectively in
Optical rotation was measured on a Rudolph Autopol IV
polarimeter. UV spectrum was taken in MeOH using a Hitachi U-
3310 spectrophotometer. IR spectrum was recorded in KBr discs
on a Nicolet FT-IR AVATAR 370 spectrophotometer. ESI-MS and
ESI-MS/MS were obtained on a Finnigan Surveyor LC-LCQ
Advantage Max spectrometer, and its conditions of the
measurements were as follows: the samples were dissolved
in methanol and were injected into the mass spectrometer by a
nature, and 2 is the first natural alkaloidal glycoside, which might
possibly be biosynthesized via O-glycosylation of 1. In addition, our
findings suggest that the chemical profile of the genus Nigella could
be characterized by the occurrence of indazole-type alkaloids,
since natural indazole-type alkaloids have only been isolated from
species of the genus Nigella to date.
Some pharmaceutically important indazoles (including bin-
darit, benzydamine, bendazac, lonidamine, granisetron) exhibit a
variety of biological activities such as anti-inflammatory (Quane
et al., 1998; Mora et al., 2012), aldose reductase inhibitors (Balfour
and Clissold, 1990), anticancer (Cosimo et al., 2003), neuronal
nitric oxide synthase inhibitor (Boulouard et al., 2007),
adrenergic receptor agonist (Harada et al., 2005) and 5-HT
receptor antagonist (Cerecetto et al., 2005; Hsu, 2010; Keating
et al., 2012). Although specifically there is not still a report on the
biological activities of naturally occurring alkaloids with a tricyclic
pyridazino[1,2-a]indazolium ring system like compounds 1 and 2,
ꢀ1
syringe-pump at 5
ml min ; the MS full scan and MS/MS scan
were carried out in positive ESI ionization mode; spray voltage
was 4.0 kV and ion transfer capillary temperature was 200 8C,
nitrogen was used as sheath and auxiliary gases with flow rates
set at 35 and 5 units, respectively; helium was used as collision
gas for MS/MS, and the normalized collision energy for MS/MS
experiments was 35%. HR-ESI-MS were obtained on a Bruker
micrOTOF-Q II spectrometer. Elemental analyses (CHN) were
b
3
-
3
1
13
performed on an Elementar Vario EL cube. H and C NMR
spectra were acquired on a Bruker Avance III 400 spectrometer
with the residual solvent signals as an internal standard (CD
3
OD
1
1
H C
d 3.307 ppm, d 49.1 ppm). H– H DQF-COSY, NOESY, HSQC
and HMBC spectra were recorded using standard Bruker
programs. CC was performed on self-packed columns with
silica gel from Qingdao Haiyang Chemical Group Co., Ltd.
(
QHCC, PR China). MPLC was carried out on a BUCHI apparatus
equipped with a C-605 pump. TLC analyses were conducted on
plates precoated with 10–40 m of silica gel from QHCC,
detected under a UV lamp at 254 nm and visualized by spraying
% phosphomolybdic acid–MeOH solution (w/v) followed by
heating.
m
6
3.2. Plant material
¨
¨
The seeds of N. glandulifera were collected from Urumuqi in
Xinjiang Uigur autonomy, China, in February 2011, and identified
by Prof. Qing-Hua Liu, Xinjiang Institute of Materia Medica. A
voucher specimen (XJ-11-0213) was deposited in Tianjin Univer-
sity of Technology, Tianjin, PR China.
3.3. Extraction and Isolation
The oil-free seeds (18 kg) of N. glandulifera were extracted three
times with 95% EtOH for 2 h under reflux and then extracted three
times with 50% EtOH for 2 h under reflux. After combination and
removal of the solvent in vacuo, the EtOH extract was then
suspended in distilled water and partitioned successively with
Fig. 3. Key COSY, HMBC, and NOE correlations of compounds 1 and 2.