A secondary metabolite amberin from Amberboa ramosa

Article abstract of DOI:10.1080/10286020.2011.647503

Amberin (1), a new sesquiterpene lactone, has been isolated from the ethyl acetate soluble fraction of Amberboa ramosa together with apigenin (2), jaseocidine (3), crysoeriol (4), and 3β, 8α-dihydroxy-11α- methyl-1αH, 5αH, 6βH, 7αH, 11βH-guai- 10(14), 4 (15)-dien-6, 12-olide (5). The structures of the isolated compounds have been elucidated by 1D and 2D NMR spectroscopy.

Full text of DOI:10.1080/10286020.2011.647503

Journal of Asian Natural Products Research
Vol. 14, No. 3, March 2012, 281–285
NOTE
A secondary metabolite amberin from Amberboa ramosa
Muhammad Ibrahima, Muhammad Imranb, Bakhat Alic, Riaz Hussainb,
Fouzia Sultana Rehmania* and Abdul Malikb
aDepartment of Chemistry, University of Karachi, Karachi 75270, Pakistan; bInternational Centre
for Chemical and Biological Sciences, HEJ Research Institute of Chemistry, University of Karachi,
Karachi 75270, Pakistan; cDepartment of Chemistry, University of Science and Technology Bannu,
Bannu 28100, Pakistan
Amberin (1), a new sesquiterpene lactone, has been isolated from the ethyl acetate
soluble fraction of Amberboa ramosa together with apigenin (2), jaseocidine (3),
crysoeriol (4), and 3b, 8a-dihydroxy-11a-methyl-1aH, 5aH, 6bH, 7aH, 11bH-guai-
10(14), 4 (15)-dien-6, 12-olide (5). The structures of the isolated compounds have been
elucidated by 1D and 2D NMR spectroscopy.
Keywords: amberin; guaianolide; Amberboa ramosa; Compositae
1. Introduction
iol (4) [6], and 3b, 8a-dihydroxy-11a-
methyl-1aH, 5aH, 6bH, 7aH, 11bH-guai-
10(14), 4 (15)-dien-6, 12-olide (5) [2] have
also been isolated and characterized.
The genus Amberboa belongs to the family
Compositae and comprises six species.
Amberboa ramosa is an annual herbaceous
plant that is mainly found in Pakistan and
India. A. ramosa has tonic, aperient,
febrifuge, deobstruent, cytotoxic, and
antibacterial properties [1]. The butyryl-
cholinesterase inhibitory activity of the
chloroform soluble fraction has also been
reported previously [2]. Literature survey
revealed that triterpenoids, flavonoids,
steroids, and sesquiterpene lactones have
previously been reported from this species
[1,3]. The chemotaxonomic and ethno-
pharmacological significance of the genus
Amberboa prompted us to reinvestigate the
constituents of A. ramosa. As a result, we
herein report the isolation and structural
elucidation of one new sesquiterpene
lactone named as amberin (1). In addition,
the previously reported compounds api-
genin (2) [4], jaseocidine (3) [5], crysoer-
2. Results and discussion
Amberin (1) was isolated as white crystals
20
with ½aꢀD þ 51.1 (c ¼ 0.02, CHCl3) and
mp 137–1398C. The HR-ESI-MS exhib-
ited a pseudomolecular ion peak at m/z
469.2068 [M 2 H]2, corresponding to the
molecular formula C23H33O10. Further
prominent peaks at m/z 308 [Mþ-162],
290 [Mþ-162-H2O], 265 [Mþ-162-
COCH3], 250 [Mþ-162-COCH3 –Me],
and 247 [Mþ –COCH3 –H2O] were
present.
The IR spectrum showed characteristic
absorption bands at 3447, 1755, 1735, and
1656 cm21 for hydroxyl, g-lactone, ester,
and olefinic functionalities, respectively.
The UV absorption maxima at 197 and
205 nm were typical of a g-lactone [6].
*Corresponding author. Email: fsrehmani@uok.edu.pk
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.647503
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282
M. Ibrahim et al.
Further spectral data showed close agree-
ment with a guaianolide-type sesquiter-
pene [3,7,8].
between the methyl protons at C-10
resonating at d 1.16 and the carbonyl
carbon at d 181.0 as shown in Figure 1. The
hydroxyl group could be assigned to C-10
based on its lower frequency shift com-
pared to unsubstituted guaianolides and
supported by HMBC experiment, in which
the methyl protons at C-14 at d 1.16 showed
2J and 3J correlations with C-10 at d 81.1,
C-9 at d 42.2, and C-1 at d 53.0.
The 13C NMR spectrum (broadband
and DEPT) showed 23 signals comprising
four methyl, three methylene, twelve
methine, and four quarternary carbons.
The low frequency region showed four
signals at d 181.0, 172.1, 144.0, and 127.1,
which could be assigned to O-acetyl,
lactone, ester, and trisubstituted olefinic
carbons. One oxygenated quaternary and
two oxygenated methine carbons resonated
at d 81.1, 75.4, and 82.3, respectively.
The position of substituents could be
confirmed by HMQC, HMBC, and COSY
experiments.
The 1H NMR spectrum showed the
signals for a trisubstituted double bond at d
5.50. It also showed signals for the
oxymethine protons at d 5.08 (ddd,
J ¼ 8.3, 6.0, 5.5 Hz) and 4.33 (dd,
J ¼ 10.0, 9.0 Hz). The latter was assigned
to the proton geminal to the lactone oxygen
atom. The larger coupling constant allowed
us to assign b and axial orientation which is
characteristic of all guaianolides of the
genus Amberboa and Ixeris [9]. It showed
1H–1H correlations with the vicinal
protons at d 2.78 (dd, J ¼ 9.0, 8.5 Hz)
and d 2.87 (ddd, J ¼ 10.0, 9.5, 8.3 Hz),
which could subsequently be assigned to
H-5 and H-7, respectively. The larger
coupling constants suggested trans-diaxial
disposition among H-5, H-6 (b), and H-7,
providing conclusive evidence for an
a-orientation of both H-5 and H-7 [9].
The coupling pattern of the proton
signals for H-1 and H-9 supported the
guaianolide structure [10]. The entire
sequence of protons attached to the
guaianolide skeleton was established by
correlation spectroscopy (COSY) and spin
decoupling experiments. Irradiation of the
H-5 proton at d 2.78 simplified the double
doublet of H-6 at d 4.33 into a doublet and
the doublet of double doublets of H-1 into
a double doublet.
The relative stereochemistry at various
chiral centers of amberin (1) was assigned
through NOESY experiments [11–13]
(Figure 2), which revealed trans/anti/cis-
fusion of the a-methyl-g-lactone moiety,
the seven member ring at C-7 and C-6, the
five member ring at C-5 and C-1. The cis-
fusion of guaianoloid is also proven by the
Irradiation of H-7 at d 2.87 simplified
the doublet of quartet at d 2.34 into a
quartet. Irradiation of H-11 at d 2.34
simplified the doublet of the methyl group
at d 1.23 into a singlet, confirming the
presence of a methyl group at C-11.
Irradiation of H-3 at d 5.50 turned the
double doublets at d 2.26 (H-2a) and 2.37
(H-2b) into doublets. The signal at 1.80
could be assigned to methyl protons at C-4
and further confirmed by 2J and 3J
correlations with C-4 (d 144.0), C-3 (d
127.1), and C-5 (d 55.0). The O-acetyl
group was assigned to C-10, on the basis of
the strong correlation in HMBC spectrum
O
O
H
H
H
H
H
O
O
H
O
O
HO
OH
HO
HO
Figure 1. Structure of amberin (1).

Journal of Asian Natural Products Research
283
O
O
O
O
H
H
H
H
H
H
H
H
O
O
H
OH
O
H
H
OH
O
H
H
O
HO
O
O
O
HO
HO
OH
Figure 3. Important HMBC correlations of
amberin (1).
HO
HO
3. Experimental
Figure 2. Important NOESY correlations of
amberin (1).
3.1 General experimental procedures
Melting points were determined on a
Gallenkemp apparatus and are uncorrected.
IR spectra were measured on a JASCO
302-A spectrophotometer with KBr cells.
UV spectra were obtained on a Hitachi UV-
3200 spectrophotometer. Optical rotations
were measured on a JASCO DIP-360
polarimeter and the 1D and 2D NMR
spectra were recorded on a Bruker AMX-
400 Spectrometer operating at 400 MHz for
1H and 100 MHz for 13C. Electron impact
(EI) mass spectra were recorded on JEOL
JMS-HX-110 and Varian MAT-311-A
mass spectrometers. The HR-ESI-MS was
recorded on a Jeol JMS 600H instrument.
Silica gel (230–400 mesh, E. Merck,
Darmstadt, Germany) was used for column
chromatography.
strong correlation between the H-1 at d
2.71 and H-5 at d 2.78. The a-orientation
of the acetate group at C-10 could also be
deduced through strong interaction in
NOESY experiment between the methyl
protons attached to C-10 resonating at d
1.16 and H-8 at d 5.08, and the methyl
group revealing its b-orientation.
The position of the hexose moiety at
C-8 could be confirmed by HMBC
correlation of the anomeric proton at d
4.54 with C-8 (d 81.1). The larger
coupling constant of the anomeric proton
allowed us to assign b-configuration to the
sugar moiety. The signals of a hexose
moiety in broadband spectra at d 98.4
(C-10), 75.5 (C-20), 78.2 (C-30), 71.8 (C-
40), 77.7 (C-50), and 62.9 (C-60) as well as
low-frequency shift of C-8 by 81.1 ppm
further revealed its presence at C-8. Acid
hydrolysis provided D-glucose that could
be identified through the sign of its optical
rotation and the retention time of its
TMS ether in gas chromatography (GC).
Thus, the structure of amberin C (1) could
be assigned as 10a-acetoxy-8a-O-(b-D-
glucopyranosyl)-1aH, 5aH, 6bH, 8bH,
7aH, 11bH, 11a-methylguaia-3-enolide
(Figure 3).
3.2 Plant material
The whole plant of A. ramosa Jafri
(Compositae) was collected in June 2002
from Karachi (Pakistan) and identified by
Dr Surraiya Khatoon, Plant Taxonomist,
Department of Botany, University of
Karachi, where a voucher specimen (no.
KU 312 b) has been deposited.
3.3 Extraction and isolation
The shade dried plant material (22 kg) was
extracted with methanol (3 £ 40 l) for

284
M. Ibrahim et al.
Table 1. 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectral data of amberin (1)
(CD3OD).
10 days at room temperature. The solvent
was evaporated under reduced pressure
and the residue (217 g) was partitioned
between n-hexane and water. The water
soluble fraction was further extracted with
chloroform, ethyl acetate, and n-butanol.
The column chromatography of the
ethyl acetate soluble fraction (90 g) over
silica gel and elution with n-hexane–ethyl
acetate in increasing order of polarity
afforded four major fractions: A (20 g) [n-
hexane–ethyl acetate (8:2)], B (13 g) [n-
hexane–ethyl acetate (6.5:3.5)], C (16 g)
[n-hexane–ethyl acetate (5.5:4.5)], and D
(20 g) [n-hexane–ethyl acetate (4:6)].
Fraction B was chromatographed over
silica gel, eluting with mixture of n-
hexane–ethyl acetate in increasing order
of polarity to afford sub-fractions BA (3 g)
[n-hexane–ethyl acetate (7:3)], BB (4.5 g)
[n-hexane–ethyl acetate (6:4)], and BC
(4 g) [n-hexane–ethyl acetate (6.5:3.5)].
The fraction BA on further chromatography
over silica gel and elution with n-hexane–
ethyl acetate (6.5:3.5) yielded compound 3
(9 mg). The chromatography of fraction BC
and elution with n-hexane–ethyl acetate
(6:4) furnished compound 4 (11.5 mg).
Fraction C was chromatographed over
silica gel and eluted with mixtures of n-
hexane–ethyl acetate to provide two sub-
fractions CA (5.4 g) [n-hexane–ethyl acet-
ate (4.5:5.5)] and CB (6.5 g) [n-hexane–
ethyl acetate (4:6)]. Fraction CB on further
chromatography over silica gel and elution
with n-hexane–ethyl acetate (7:3)
afforded compound 5 (10 mg).
Positions
dC
dH multiplicity (J in Hz)
1
2
53.0
34.0
2.71 ddd (9.0, 8.5, 8.0)
2.37 dd (8.0, 7.0)
2.26 dd (9.0, 5.0)
5.50 m
3
4
5
6
7
8
9
127.1
144.0
55.0
82.3
52.3
81.1
42.8
–
2.78 dd (9.0, 8.5)
4.33 dd (10.0, 9.0)
2.87 ddd (10.0, 9.5, 8.3)
5.08 ddd (8.3, 6.0, 5.5)
1.87 dd (12.3, 5.5)
2.39 dd (12.3, 6.0)
–
10
11
12
13
14
15
16
17
10
75.4
42.1
172.1
15.7
26.1
17.7
181.0
21.3
98.4
75.5
78.2
71.8
77.7
62.9
2.34 dq (9.5, 5.8)
–
1.23 d (6.9)
1.16 s
1.80 s
–
2.06 s
4.54 d (7.5)
3.28 m
3.38 m
20
30
40
3.17 m
3.23 m
3.82 dd (2.1, 12.0)
3.6 dd (6.0, 12.0)
50
60
n
max cm21: 1735, 3447, 1755, and 1656.
UV lmax: 197 and 215 nm. H and 13C
spectral data, see Table 1. EI-MS: m/z
470 [M]þ, 308, 290, 265, 250, and 247.
HR-ESI-MS: m/z 469.2068 [M 2 H]2
(calcd for C23H33O10, 469.2074).
1
3.4 Acid hydrolysis of amberin (1)
Fraction D was chromatographed over
silica gel and eluted with mixtures of ethyl
acetate–methanol in increasing order of
polarity to obtain compound 1 [ethyl
acetate–methanol (9:1)] (11 mg) and
compound 2 [ethyl acetate–methanol
(7:3)] (13 mg).
Amberin (1) (2 mg) in MeOH (3 ml)
containing 1 N HCl (3 ml) was refluxed
for 4 h, concentrated under reduced press-
ure, diluted with water (7 ml) and extracted
with ethyl acetate. The residue recovered
from the organic phase was a mixture of
aglycone products which could not be
worked up due to paucity of material. The
aqueous phase was concentrated and
D-glucose was identified by its optical
3.3.1 Amberin (1)
20
White crystals, ½aꢀD þ 51.1 (c 0.02,
20
CHCl3), mp 137–1398C. IR (KBr)
rotation ½aꢀD ¼ þ50.6 and by comparing

Journal of Asian Natural Products Research
285
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