A New Alkaloid from Isatis costata
Turk J Chem
31 (2007) , 443 – 447.
c TÜBİTAK
A New Alkaloid from Isatis costata
Itrat FATIMA1 , Itrat ANIS1 , Ijaz AHMAD2 , Abdul MALIK1∗, Nighat AFZA3
1
International Center for Chemıcal Sciences, Hej Research Institute of Chemistry,
University of Karachi, 75270, Karachi-PAKISTAN
e-mail: abdul.malik@ıccs.edu
2
Medicinal Botanic Centre, Pcsir Labs. Complex Peshawar, Peshawar, N.W.F.P, PAKISTAN
3
Pharmaceutical Research Centre, Pcsir Labs. Complex, 75270, Karachi-PAKISTAN
Received 21.09.2006
A new alkaloid has been isolated from Isatis costata C.A.Mey. and assigned structure 1 on the
basis of spectroscopic data including 1D and 2D NMR techniques. Methyl 2-acetoamidobenzoate (2),
β-sitosterol (3), and ursolic acid (4) were also isolated for the first time from this species.
Key Words: Isatis costata, Brassicaceae, 2D NMR, structure elucidation.
Introduction
The genus Isatis, belonging to the family Brassicaceae, comprises 50 species mainly distributed in the IranoTuranian region. In Pakistan, it is represented by 7 species.1 Isatis tinctoria, also known as woad, is a
common plant cultivated for centuries to produce the blue dye indigo. Nowadays, woad is also used in
Chinese folk and modern medicine.2 “Ban-Lan-Gen” is one of the most commonly used traditional Chinese
medicines for antipyretic, anti-inflammatory, antiviral, and detoxifying purposes. Its original source was
considered to be the dried roots of 3 plants, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia.3,4
Now the roots of Isatis indigotica have been identified as the main source of “Ban-Lan-Gen” and recorded
in Chinese Pharmacopoeia (1990 edn).5 The ethnopharmacological importance of the genus Isatis prompted
us to investigate the chemical constituents of Isatis costata C.A.Mey., which is an annual or biennial herb
found in northern Pakistan. Previously 2 oxindole alkaloids have been reported from this species.6 Herein we
report the isolation and structural elucidation of a new alkaloid (1) along with methyl 2-acetoamidobenzoate
(2), β-sitosterol (3), and ursolic acid (4), which are reported for the first time from this species.
Experimental
General experimental procedures
Optical rotations were recorded on a JASCO DIP-360 digital polarimeter. IR spectra were measured on a
JASCO 302-A spectrophotometer in CHCl3 . UV spectra were obtained on a Hitachi UV-3200 spectropho∗ Corresponding
author
443
A New Alkaloid from Isatis costata, I. FATIMA, et al.,
tometer. NMR spectra were run on a Bruker instrument. Chemical shifts δ are shown in ppm relative to
TMS as internal standard and coupling constants J are described in Hz. EI, FAB, and HREI-MS were
recorded on JEOL JMS-HX-110 and JMS-DA-500 mass spectrometers. Silica gel 230-400 mesh (E. Merck)
was used for column chromatography. Silica gel plates (Si 60 F254 , E. Merck) were used for TLC.
Plant material
The whole plant material was collected in April 2004 from the Swat Valley in the Northwestern Frontier
province of Pakistan and identified as Isatis costata C.A.Mey. by Dr. Ghosia Lutfullah, Center of Biotechnology, University of Peshawar, Pakistan. A voucher specimen (BPU-105) is deposited in the herbarium of
the Department of Botany, University of Peshawar, Peshawar, Pakistan.
Extraction and isolation
The shade-dried whole plant (30 kg) was chopped and extracted 3 times with EtOH (60 L) at room
temperature for 96 h. The ethanolic extract was evaporated in vacuo to give a dark greenish residue (400
g), which was partitioned between n-hexane and water. The aqueous fraction was further extracted with
EtOAc and n-BuOH. The EtOAc fraction (12 g) was subjected to column chromatography eluting with nhexane-EtOAc in increasing order of polarity to give 3 fractions. The silica gel column chromatography of the
fraction obtained from n-hexane-EtOAc (7:3) showed 2 major spots on TLC, which on further purification by
column chromatography over silica gel using n-hexane-EtOAc (8:2) as eluent yielded compounds 1 (11 mg)
and 2 (15 mg). On the other hand, the eluent obtained from n-hexane-EtOAc (4:6) was rechromatographed
over silica gel to afford 2 successive fractions using solvent system n-hexane-EtOAc (8:2-5:5). The second
fraction was rechromatographed and eluted with n-hexane-EtOAc (7:3) to afford compounds 3 (9 mg) and
4 (11 mg) from the top and the tail fractions, respectively.
Compound 1: White amorphous solid, mp 110-112 ◦ C; UV (MeOH) λmax nm (log ε) = 205 (4.08),
288 (3.25), 175 (4.12). IR (KBr) νmax = 3440, 1717, 1690, 1680, 1615, 1500, 1455 cm−1 . EI-MS, m/z
(%) = 331 [M+ ] (47), 224 (100), 120 (72), 119 (40), 91 (45), 77 (85). HRFABMS m/z 331.1208 (calc. for
C21 H17 NO3 331.1205). Complete assignments of 13 C- and 1 H-NMR signals for 1 are described in the Table.
Important HMBC correlations are illustrated in the Figure.
Methyl 2-acetoamidobenzoate (2): White amorphous solid, mp 101 ◦ C; IR (KBr) νmax = 1690,
1680, 1615, 1500, 1455, 3440 cm−1 . HRFABMS m/z 193.0738, (calc. for C10 H11 NO3 193.0735). EI-MS, 13 Cand 1 H-NMR data were identical to those reported in the literature.7
β-Sitosterol (3): Colorless crystals, mp 258-262 ◦ C; IR (KBr), νmax = 3450, 3050, 1650, 81 cm−1 ;
EI-MS and NMR data were identical to those reported in the literature.8
Ursolic acid (4): Colorless crystals, mp 110-112 ◦ C; IR (KBr), νmax = 3510, 3050, 1697, 1635, 820
cm−1 . EI-MS and NMR data were identical to those reported in the literature.9
Results and Discussion
The ethanolic extract of shade-dried whole plant (30 kg) of Isatis costata C.A.Mey. was partitioned in
between n-hexane, EtOAc, n-BuOH, and H2 O. As a result of a series of column chromatographic techniques
compounds 1-4 were isolated from the EtOAc fraction as described in the Experimental part.
444
A New Alkaloid from Isatis costata, I. FATIMA, et al.,
Table.
13
C- (125 MHz) and 1 H-NMR (500 MHz) spectral data (CD3 OD) for compound 1.
13
1
1
C
(δ, ppm)
120.8
2
139.3
-
3
127.5
7.78, dd, J = 8.3,0.8 Hz
4
129.3
7.55, ddd, J = 8.3, 7.6, 1.5 Hz
5
122.9
7.09, ddd, J = 8.4, 7.6, 0.8 Hz
6
121.3
8.78, dd, J = 8.4, 1.5 Hz
1
137.6
-
2
128.6
7.35, m
3
129.1
7.35, m
4
131.3
7.35, m
5
129.1
7.35, m
6
128.6
7.35, m
7
169.1
-
8
Position
H
(Pδ, ppm)
-
43.6
4.54, d, J = 5.5 Hz
134.4
-
2
127.4
7.98, dd, J = 7.5, 1.4 Hz
3
128.5
7.48, m
4
132.4
7.46, m
128.5
7.48, m
6
127.4
7.98, dd, J = 7.5, 1.4 Hz
7
165.9
-
N–H
-
12.1
1
5
2`
O
6
5
4
3
8` 1`
C 7`
NH
1
2
3`
4`
5`
6`
O
C 1``
2``
3``
4``
O 7``
6``
5``
Compound 1
445
A New Alkaloid from Isatis costata, I. FATIMA, et al.,
H
H
O
H
H
C
NH
H
O
H
O
H
C
H
H
H
Figure. The significant long range correlations detected in the HMBC spectrum of 1.
Compound 1 was isolated as a white amorphous solid and gave positive test results with Dragendorff’s
reagent for nitrogenous compounds. The molecular ion peak at m/z 331.1201 in the HR-EIMS indicated its
molecular fo (...truncated)
