Structural determination of abutilins A and B, new flavonoids from Abutilon pakistanicum, by 1D and 2D NMR spectroscopy

Article abstract of DOI:10.1002/mrc.2546

Two new flavonoids, abutilin A and B, were isolated from the chloroform soluble fraction of Abutilon pakistanicum and their structures assigned from ¹H and ¹³C NMR spectra, DEPT and by 2D COSY, HMQC and HMBC experiments. Ferulic acid (3), (E)-cinnamic acid (4), 5-hydroxy-4′,6,7,8- tetramethoxyflavone (5), kaempferol (6), luteolin (7) and luteolin 7-O-β-D-glucopyranoside (8) have also been reported from this species. Copyright

Full text of DOI:10.1002/mrc.2546

Spectral Assignments and Reference Data
Received: 5 August 2009
Revised: 21 October 2009
Accepted: 21 October 2009
Published online in Wiley Interscience: 20 November 2009
(www.interscience.com) DOI 10.1002/mrc.2546
Structural determination of abutilins A and B,
new flavonoids from Abutilon pakistanicum,
by 1D and 2D NMR spectroscopy
Bakhat Ali, Muhammad Imran, Riaz Hussain, Zaheer Ahmed
and Abdul Malik∗
Two new flavonoids, abutilin A and B, were isolated from the chloroform soluble fraction of Abutilon pakistanicum and their
structures assigned from 1H and 13C NMR spectra, DEPT and by 2D COSY, HMQC and HMBC experiments. Ferulic acid (3), (E)-
cinnamicacid(4), 5-hydroxy-4ꢀ,6,7,8-tetramethoxyflavone(5), kaempferol(6), luteolin(7) andluteolin7-O-β-D-glucopyranoside
c
(8) have also been reported from this species. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Keywords: NMR; 1D/2D NMR; flavonoids; Abutilon pakistanicum; Malvaceae; abutilin A; abutilin B
•
Introduction
group at C-5 of a flavonoid.[16] On the other hand, addition of
NaOAc resulted in a bathochromic shift of 11 nm, suggesting the
presence of free hydroxyl group at C-7. The IR spectrum showed
the presence of hydroxyl group (3330–3445 cm−1), ester carbonyl
(1675 cm−1), conjugated carbonyl (1660 cm−1) and aromatic
moieties (1535 and 1500 cm−1). The molecular formula was
deduced as C24H24O12 through high-resolution electron impact
mass spectroscopy (HR-EI-MS) which showed the M+ peak at m/z
504.1263 (calculated for C24H24O12, 504.1268). The broadband
(BB) and DEPT 13C NMR spectra displayed 24 signals comprising
10 quaternary, 11 methine, 1 methylene and 2 methyl carbons
(Table 1).
The genus Abutilon, which belongs to the family Malvaceae,
comprises 150 species and is distributed in subtropical regions
of Asia and other parts of the world in the form of perennial
herbs and shrubs and rarely as small trees. Generally the
leaves, roots and stems of Abutilon species contain considerable
amounts of mucilage because of which these are used for the
treatment of rheumatism and as demulcents and diuretics.[1–3]
The ethnopharmacological and chemotaxonomic importance of
the genus Abutilon led us to investigate the chemical constituents
of one of its species, namely Abutilon pakistanicum. The literature
survey revealed that only one steroid and three triterpenes have
so far been reported from this species.[4–6] We now report the
isolation and structural elucidation of two new flavonoids named
abutilins A (1) and B (2) based on spectroscopic techniques
including 1D and 2D NMR. In addition, ferulic acid (3),[7]
(E)-cinnamic acid (4),[8] 5-hydroxy-,4ꢁ,6,7,8-tetramethoxyflavone
(5),[9] kaempferol (6),[10,11] luteolin (7)[12] and luteolin 7-O-β-D-
glucopyranoside (8)[13] have also been isolated for the first time
from this species.
TheIHNMRspectrumof1showedacharacteristiclow-frequency
signal at δ 13.6 assignable to the chelated hydroxyl group at C-5
of a flavonoid. Two meta-coupled aromatic protons at δ 6.74 (1H,
d, J = 2.1 Hz) and δ 6.81 (1H, d, J = 2.1 Hz) were diagnostic for
a C-5 and C-7 oxygenated ring A. The 1H NMR spectrum showed
three further aromatic protons of ring B forming an ABX system
at δ 7.16 (1H, d, J = 8.8 Hz), 7.71 (1H, dd, J = 8.8, 2.1 Hz) and
7.55 (1H, d, J = 2.1 Hz). The signal of H-3 resonated at δ 6.96 (s,
1H), and one methoxyl group was observed at 3.75 (s, 3H). The
presence of an acetylated hexose moiety in β configuration was
evident by the signals of an anomeric proton at δ 5.74 (1H, d,
J = 7.6 Hz) and a methyl singlet at δ 1.95 (3 H) due to the acetyl
moiety. Further signals of oxymethines and oxymethylene of the
sugar moiety were observed at δ 4.77 (1 H, m, H-2ꢁꢁ), 4.25 (1 H,
m, H-3ꢁꢁ), 4.14 (1 H, m, H-4ꢁꢁ), 4.28 (1 H, m, H-5ꢁꢁ), 4.96 (1 H, dd,
J = 11.2, 2.6 H-6ꢁꢁ a) and 4.39 (1 H, dd, J = 11.2, 2.8, H-6ꢁꢁ b).
EI-MS spectra showed a peak at m/z 300 resulting from the loss of
aceylated hexose moiety, and further retro Diels–Alder fragments
Results and Discussion
The methanolic extract of the whole plant was divided into n-
hexane, chloroform, ethyl acetate, n-butanol and water soluble
fractions. Column chromatography (CC) of the chloroform soluble
fraction provided compounds 1–8. Their structures were estab-
lished by UV, IR, MS and NMR spectroscopy. Both abutilins A (1)
and B (2) were isolated as yellow amorphous powder, and they
gave violet coloration with ferric chloride for a phenolic moiety
and were recognized as flavonoid glycosides from their positive
tests with Molish and Shinoda reagents.[14]
The UV spectrum of abutilins A (1) was characteristic of
flavonoidal glycoside showing absorptions at λmax 206, 275 and
315 nm.[15] On addition of AlCl3/HCl, it showed a bathochromic
shift of 40 nm, suggesting the presence of chelated hydroxyl
∗
Correspondence to: Abdul Malik, International Center for Chemical and
Biological Sciences, H. E. J. Research Institute of Chemistry, University of
Karachi, Karachi 75270, Pakistan. E-mail: abdul.malik@iccs.edu
International Center for Chemical and Biological Sciences, H. E. J. Research
Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan
c
Magn. Reson. Chem. 2010, 48, 159–163
Copyright ꢀ 2009 John Wiley & Sons, Ltd.

B. Ali et al.
Table 1. 1H, 13C NMR and HMBC correlations of compound 1 (C5D5N)
Table 2. 1H, 13C NMR and HMBC correlations of compound 2 (C5D5N)
Position
13C-NMR
1H-NMR
HMBC
Position
13C-NMR
1H-NMR
HMBC
2
3
4
5
6
163.8
105.1
182.6
158.5
100.1
–
–
2
157.6
134.4
178.7
157.8
99.9
–
–
6.96 (1H, s)
2, 4, 10, 1ꢁ
3
–
–
–
–
–
–
4
–
–
5
–
–
6.74 (1H, d,
5, 7, 8, 10
6
6.67 (1H, d, J = 2.0 Hz)
5, 7, 8, 10
J = 2.1 Hz)
7
166.0
94.6
–
–
7
8
165.9
95.0
–
–
8
6.65 (1H, d, J = 2.0 Hz)
6, 7, 9
6.81 (1H, d,
6, 7, 9
9
162.7
105.2
121.9
130.6
116.0
161.7
–
–
–
J = 2.1 Hz)
10
1ꢁ
2ꢁ, 6ꢁ
3ꢁ, 5ꢁ
4ꢁ
–
–
9
163.0
104.1
–
–
–
–
–
2, 3ꢁ, 4ꢁ, 5ꢁ
2ꢁ, 4ꢁ, 6ꢁ
–
10
5-OH
1ꢁ
–
–
8.42 (2H, d, J = 8.7 Hz)
13.6
–
–
7.21(2H, d, J = 8.7 Hz)
125.6
115.9
–
–
2ꢁ
7.55 (1H, d,
1ꢁ, 3ꢁ, 4ꢁ
3-OH
5ꢁ-ꢁOH
1ꢁꢁ
12.34
–
J = 2.1 Hz)
–
13.18
6.21 (1H, d, J = 7.5 Hz)
4.21 (m)
–
3ꢁ
4ꢁ
5ꢁ
150.8
150.1
110.6
–
–
–
–
104.1
76.0
4ꢁ, 2ꢁꢁ, 5ꢁꢁ
1ꢁꢁ, 4ꢁꢁ
1ꢁꢁ, 2ꢁꢁ, 5ꢁꢁ
2ꢁꢁ, 5ꢁꢁ
4ꢁꢁ
2ꢁꢁ
7.16 (1H, d,
3ꢁ, 4ꢁ, 6ꢁ
3ꢁꢁ
71.3
4.18 (m)
J = 8.8 Hz)
4ꢁꢁ
78.4
4.82 (m)
6ꢁ
120.3
101.7
7.71 (1H, dd,
1ꢁ, 2ꢁ, 5ꢁ
4ꢁ 2ꢁꢁ, 5ꢁꢁ
5 a
5ꢁꢁb
1ꢁꢁꢁ
2ꢁꢁꢁ
3ꢁꢁꢁ
4ꢁꢁꢁ
5ꢁꢁꢁ, 9ꢁꢁꢁ
6ꢁꢁꢁ, 8ꢁꢁꢁ
7ꢁꢁꢁ
64.2
4.95 (1H, dd, J = 9.8, 2.0 Hz)
4.38 (1H, dd, J = 9.8, 4.1 Hz)
–
J = 8.8, 2.1 Hz)
1ꢁꢁ
5.74 (1H, d,
J = 7.6 Hz)
167.2
114.9
145.1
126.5
131.8
116.7
161.3
–
2ꢁꢁ
74.8
76.6
71.1
78.2
63.4
4.77 (m)
4.25 (m)
4.14 (m)
4.28 (m)
1ꢁꢁ, 2ꢁꢁ-OCOCH3
1ꢁꢁ, 2ꢁꢁ, 5ꢁꢁ
2ꢁꢁ, 5ꢁꢁ, 6ꢁꢁ
1ꢁꢁ, 6ꢁꢁ
6.48 (1H, d, J = 15.8 Hz)
7.82 (1H, d, J = 15.8 Hz)
–
3ꢁꢁꢁ, 4ꢁꢁꢁ
1ꢁꢁꢁ, 5ꢁꢁꢁ, 9ꢁꢁꢁ
–
3ꢁꢁ
4ꢁꢁ
5ꢁꢁ
7.48 (2H, d, J = 8.6 Hz)
7.13 (2H, d, J = 8.6 Hz)
–
4ꢁꢁꢁ, 6ꢁꢁꢁ, 7ꢁꢁꢁ
4ꢁꢁꢁ, 5ꢁꢁꢁ, 9ꢁꢁꢁ
–
6ꢁꢁa
4.96 (1H, dd,
5ꢁꢁ
J = 11.2, 2.6 Hz)
6ꢁꢁb
4.39 (1H, dd,
J = 11.2, 2.8 Hz)
2ꢁꢁ-OCOCH3
2ꢁꢁ-OCOCH3
3ꢁ-OCH3
170.5
20.6
56.0
–
1.95 (3H, s)
3.75 (3H, s)
–
2ꢁꢁ-OCOCH3
3ꢁ
1
and H-2ꢁꢁ. The HMBC (Table 1), HMQC and H–1H COSY spectra
wereincompleteagreementtotheassignedstructureofabutilinA
(1) as 5,7,4ꢁ trihydroxy-3ꢁ-methoxy flavone-4ꢁ-O-β-D-(2ꢁꢁ-O-acetyl)
glucopyranoside (Fig. 1).
The molecular formula of abutilin B (2) was deduced from
the positive mode high-resolution fast atom bombardment mass
spectrometry (HR-FAB-MS), giving a pseudomolecular ion peak
[M + H]+ at m/z 565.1342 (calculated for 565.1347), consistent
with the molecular formula C29H25O12. It was further confirmed
by the 13C NMR (BB and DEPT) spectra, which showed 29 signals
comprising 1 methylene, 16 methine and 12 quaternary carbons
(Table 2). The UV spectrum was characteristic of flavonoidal
glycoside, showing absorptions at λmax 208, 265 and 321 nm.
On addition of AlCl3/HCl, it showed significant bathochromic
shifts due to chelated phenolic groups.[16] The IR spectrum
displayed the signal for hydroxyl group (3320–3415 cm−1),
ester carbonyl (1684 cm−1), conjugated carbonyl (1665 cm−1),
olefinic bond (1645 cm−1) and aromatic moieties (1540 and
1505 cm−1).
at m/z 152 and m/z 148 confirmed the presence of methoxyl and
hydroxyl groups in ring B, and two hydroxyl groups in ring A,
respectively.
In BB and DEPT (135 and 90) 13C NMR spectra, the signals at δ
163.8, 105.1, 182.6, 163.0 and 104.1 were typical of C-2, C-3, C-4,
C-9 and C-10 of a flavonol moiety.[17] The position of methoxyl
group was not only authenticated through HMBC but also through
nuclear Overhausser effect (NOE) between methoxyl protons at
δ 3.75 and H-2ꢁ. The 1H and 13C NMR data of the flavonoid
skeleton were in complete agreement to those of chrysoeriol.[18]
Abutilin A (1) is, therefore, the glycoside of chrysoeriol. Acid
hydrolysis provided chrysoeriol (mp 330–331 ◦C) and the sugar
moiety, the latter being confirmed as D-glucose through the sign
of its optical rotation ([α]D + 52.8) and comparative thin-layer
chromatography (Co-TLC) with an authentic sample. The position
of glucose moiety was established at C-4ꢁ by HMBC experiments
in which the anomeric proton at δ 5.74 showed 3J correlation
with C-4ꢁ at δ 150.1. The low frequency shift of H-2ꢁꢁ allowed us to
assign the acetyl moiety to this position. It was confirmed through
HMBC experiments in which the ester carbonyl carbon at δ 170.5
I
The H NMR spectrum of 2 showed low frequency signals at
δ 12.34 and 13.18 assignable to the chelated hydroxyl groups at
C-3 and C-5 of a flavonoid moiety. Two meta-coupled doublets
(J = 2.0 Hz), each integrating for one proton at δ 6.67 and 6.65,
were diagnostic for a C-5 and C-7 oxygenated ring A. It further
showed the presence of four aromatic protons of para-substituted
ring B showing AAꢁXXꢁ pattern at δ 8.42 (d, J = 8.7 Hz) and
7.21 (d, J = 8.7 Hz), respectively. The presence of (E)-p-coumaroyl
moiety could be inferred from the trans-olefinic protons at δ 7.82
2
3
showed J and J correlations with acetyl methyl at δ 1.95 and
H-2ꢁꢁ at δ 4.77. This could further be confirmed through H–1H
1
COSY, which showed correlation between the anomeric proton
c
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Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2010, 48, 159–163

Structural determination of abutilins A and B by 1D and 2D NMR spectroscopy
H3C
H3C
O
O
O
1′′
2′′
5′′
3′′
3′
O
OH
2′
1′
O
4′
5′
4′′
OH
8
OH
6′′
O
O
HO
2
9
7
6
6`
3
10
4
5
OH
1
6′′′
HO
1′′
HO
3′
6′
7′′′
2′′
5′′
5′′′
4′′′
O
3′′
2′
1′
4′
5′
O
OH
2′′′
8
8′′′
4′′
1′′′
O
2
O
9
9′′′
7
3′′′
OH
3
O
6
10
4
5
OH
OH
O
2
Figure 1. Structures of abutilins A (1) and B (2).
and δ 6.48 (d, J = 15.8 Hz)[19] as well as protrons of p-hydroxy
phenyl ring showing another AAꢁXXꢁ pattern at 7.48 (d, J = 8.6 Hz)
and 7.13 (d, J = 8.6 Hz), respectively. The presence of a pentose
moiety was revealed by the signal of anomeric proton at δ 6.21
and further signals of oxymethine and oxymethylene protons at
δ 4.21 (m, H-2ꢁꢁ), 4.18 (m, H-3ꢁꢁ), 4.82 (m, H-4ꢁꢁ), 4.95 (dd, J = 9.8,
2.0, H-5ꢁꢁ a) and 4.38 (dd, J = 9.8, 4.1, H-5ꢁꢁ b). The 13C NMR
signals at δ 157.6, 134.4, 178.7, 162.7 and 105.2 were typical of
C-2, C-3, C-4, C-9 and C-10 of a flavonol moiety. The anomeric
carbon was observed at δ 104.1 and further signals of the pentose
moiety were observed at δ 76.0 (C-2ꢁꢁ), 71.3 (C-3ꢁꢁ), 78.4 (C-4ꢁꢁ)
and 64.2 (C-5ꢁꢁ). The 13C NMR spectrum further showed signals of
ester carbonyl at δ 167.2 and olefinic carbons at 114.9 and 145.1
along with signals of benzene ring moiety at δ 126.5, 131.8, 116.7
and 161.3.
illustrated in Table 2 are in complete agreement with the assigned
structure of abutilin B (2), as 7-O-(E)-p-coumaroyl kaempferol
4ꢁ-O-α-L-arabinopyranoside (Fig. 1).
Experimental
Methods
Column chromatography was carried out using silica gel (230–400
mesh, E. Merck, Darmstadt, Germany). TLC was performed on
precoated silica gel G-25-UV254 plates (20 × 20 cm, E. Merck,
Darmstadt, Germany) and detection was done at 254 nm, and
by spraying with ceric sulfate in 10% H2SO4. Optical rotations
were measured on a JASCO DIP-360 polarimeter. UV spectra
were recorded on a Hitachi UV-3200 spectrophotometer. The
IR spectra were recorded on Jasco 302-A spectrometer. Mass
spectra (EI and HR-EI-MS) were measured in the electron impact
mode on Finnigan MAT 12 or MAT 312 spectrometers and
ions are given in m/z (%). Positive mode HR-FAB-MS was
recorded on a Jeol JMS-DA-500 mass spectrometer. Melting
points were determined on a Gallenkemp apparatus and are
uncorrected.
The key to the structure of abutilin A (2) was provided by
the acid hydrolysis, which afforded, besides the sugar moiety,
a mixture of aglycones identified as (E)-p-coumaric acid and
kaempferol.[10,11] The sugar could be identified as L-arabinose
20
through Co-TLC and sign of its optical rotation [α]D + 102.1.
The larger coupling constant of the anomeric proton (J = 7.5 Hz)
allowed us to assign α configuration to the pentose moiety.
This was also confirmed by EI-MS, which showed fragments
at m/z 432 [M+ − p-coumaroyl] and 286 [M+ − p-coumaroyl
− L-arabinose]. In addition, the presence of retro Diels–Alder
fragments at m/z 152 and m/z 134 confirmed the presence of
one hydroxyl and (E)-p-coumaroyl moiety in ring A. The remaining
problem was to locate the point of attachment of the sugar
moiety. The HMBC experiments showed 3J correlation of the
anomeric proton at δ 6.21 with C-4ꢁ (161.7), C-3ꢁꢁꢁ (71.3), C-5ꢁꢁ
(64.2) and 2J correlations with C-2ꢁꢁ (76.0). This allowed us to
assign the sugar moiety at C-4ꢁ; therefore the (E)-p-coumaroyl
moiety must be at C-7. The important HMBC correlations
NMR spectra
The 1D and 2D NMR spectra were recorded on a Bruker AMX-400
spectrometer in C5D5N operating at 400 MHz for 1H and 100 MHz
for 13C. Chemical shifts are in ppm (δ), relative to tetramethylsilane
as an internal standard, and scalar couplings are reported in hertz.
The pulse conditions were as follows: for the 1H NMR spectra,
observation frequency 400 MHz, acquisition time (AQ) = 2.732 s,
number of scans (NS) = 128, number of dummy scans (DS) = 0,
relaxation delay (RD) = 1.0 s, 90◦ pulse width = 5.40 µs, spectral
c
Magn. Reson. Chem. 2010, 48, 159–163
Copyright ꢀ 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

B. Ali et al.
MeOH
width (SW) = 7183.91 Hz, line broadening (LB) = 0.3 Hz and
fourier transform (FT) size = 32 768; for the 13C NMR spectrum,
observation frequency 100.613 MHz, AQ = 0.654 s, NS = 3000,
DS = 2, RD = 1.5 s, SW = 25075.64 Hz, FT size = 32 768, LB
= 1.5 Hz; for the DEPT 135 spectrum, observation frequency =
100.632 MHz, AQ = 1.0879476 s, NS = 10 823, DS = 2, RD = 1.5 s,
SW = 15060.241 Hz, LB = 1.00 Hz; DEPT 90 spectrum, observation
frequency = 100.618 MHz, AQ = 1.0879476 s, NS = 1029, DS = 2,
RD = 1.5 s, RG = 32 768, SW = 15060.241 Hz, LB = 1.00 Hz; the
COSY 45◦ spectra were recorded at 400 MHz at SW of 4810 Hz
in F2 3065 and F1 in the 3082 domain. The AQ was 0.14 s, the
spectra were acquired with 1K data points in F2 with 32 transients,
8 DS and 256 experiments. For the NOESY experiments the SWs
of 2904 for F2 and F1 domains were recorded. The AQ was 0.15 s,
1025 × 256 data points, 128 transients and 500 ms mixing time.
The long range 1H–13C correlations (HMBC) spectra resulted from
1024 × 256 data matrix with 16 scans per t1 increments. Spectral
width of 25 658 Hz in F1 and 3412 Hz in F2 domain were used.
The AQ was 0.14 s, the delays were set to 3.45 ms (1/2J (C, H)) and
65 ms (corresponding to an average 1/nJ (C, H) of 7.7 Hz) and the
recycle time was 1.44 s. Fourier transform was done on a 2K ×
1K data matrix. The HMQC spectra was collected in the t1 domain
in 256 experiments with 2K data points and SWs of 3375 and
20 785 in the F2 and F1 dimensions, respectively. The relaxation
delay D1 was set to 1.5 s and D2 was empirically optimized to
3.5 ms.
Compound (1): Yellow amorphous powder, UV λmax
(log
KBr
ε) nm: 206 (2.5), 275 (1.4), 315 (3.8); IR νmax cm−1: 3330–3445
(OH), 1675 (ester CO), 1660 (CO), 1535, 1500 (aromatic moieties);
25
[α]D −75.5 (c = 0.1, MeOH); EI-MS (70 e/v) (rel.int %) 504 (15),
300 (100), 285 (55), 272 (24), 257 (15), 229 (36), 162 (28), 152
(35), 148 (50), 133 (35), 114 (21), 1.5 (20). HR-EI-MS m/z 504.1263
(calculated for C24H24O12, 504.1268). For 1H, 13C NMR and HMBC
data see Table 1.
MeOH
Compound (2): Yellow amorphous powder, UV λmax
(log
KBr
ε) nm: 208 (2.9), 265 (2.2), 321 (4.5); IR νmax cm−1: 3320–3415
(OH), 1684 (ester CO), 1665 (CO), 1645 (C C), 1540, 1505 (aromatic
25
moieties); [α]D −104.5 (c = 0.05, MeOH); EI-MS (70 e/v) (rel.int
%) 432 (22), 404 (46), 339 (23), 298 (15), 286 (100), 257 (75), 232
(29), 213 (35), 209 (81), 164 (76), 152 (32), 150 (16), 134 (24) 85
(30), 60 (35). HR-FAB-MS (positive mode) m/z: 565.1342 [M + H]+
(calculated for C29H25O12, 565.1347); For 1H, 13C NMR and HMBC
data see Table 2.
Acid hydrolysis of 1 and 2
Abutilin A (1) (8 mg) in 10% HCl was refluxed for 40 min. The
cooled reaction extracted with ethyl acetate. The EtOAc fraction
crystallized from methanol, and was identified as chrysoeriol (mp
330–331 ◦C) through comparison of physical and spectral data
with those reported in the literature.[18] The aqueous phase
was concentrated and the sugar was identified as D-glucose
by the sign of its optical rotation ([α]D + 52.8) and Co-TLC
with an authentic sample of D-glucose using the solvent system
n-BuOH–EtOAc–HOAc–H2O (12 : 2 : 2 : 2). TLC was run three times
in the same direction and spots were visualized with aniline
phthalate reagent. It was further confirmed by comparing
the retention time of TMS ether with standard sample in
GC.[20,21]
Plant material
The whole plant of A. pakistanicum Jafri and Ali (8 kg) was
collected from Karachi and identified by the Plant Taxonomist,
Department of Botany, University of Karachi, where a voucher
specimen (No. 697 KUH) has been deposited.
AcidhydrolysisofabutilinB(2)undersimilarreactionconditions
yielded a mixture of aglycones which could be separated through
preparative TLC and identified as kaempferol (mp 277–279 ◦C)
and (E)-p-coumaric acid (mp 211–213 ◦C), respectively. The
sugar was identified as L-arabinose by Co-TLC using solvent
system CHCl3: MeOH (7 : 3) and the sign of its optical rotation
Extraction and isolation
The freshly collected whole plant material of A.pakistanicum (8 Kg)
was cut into small pieces and extracted with MeOH (3 × 20 l). The
combinedmethanolicextractwasevaporatedunderreducedpres-
sure to yield a residue (350 g), which was divided into n-hexane
(65 g), CHCl3 (75 g), EtOAc (35 g), n-BuOH (90 g) and water (60 g)
soluble fractions. The chloroform soluble fraction (75 g) was sub-
jected to CC over silica gel eluting with n-hexane–CHCl3, CHCl3
and CHCl3 –MeOH in increasing order of polarity. The fractions
that were obtained from n-hexane–chloroform (3.5 : 6.5) were
combined and rechromatographed over silica gel eluting with
n-hexane–chloroform (3.8 : 6.2) to afford compound 3 (25 mg)
and compound 4 (25 mg). The fractions that were obtained
from n-hexane–chloroform (1.0 : 9.0) were combined and rechro-
matographed over silica gel eluting with n-hexane–chloroform
(1.5 : 8.5) to afford compound 5 (25 mg). The fractions that were
obtained from chloroform–methanol (9.0 : 1.0) were combined
and rechromatographed over silica gel eluting with chloro-
form–methanol in increasing order of polarity. The fractions ob-
tained from CHCl3 –MeOH (9.5 : 0.5) were subjected to preparative
TLC (CHCl3 –MeOH, 8.5 : 1.5) to afford compounds 6 (17 mg) and
7 (14 mg), respectively. The fractions which were obtained from
CHCl3 –MeOH (9.0 : 1.0) were combined and rechromatographed
oversilicagelelutingwithCHCl3 –MeOH(9.3 : 0.7)toaffordabutilin
A (1) (18 mg) and compound 8 (25 mg). The fractions that were
obtained from CHCl3 –MeOH (8.0 : 2.0) were combined and sub-
jected to preparative TLC over silica gel (CHCl3 –MeOH; 7.2 : 2.8) to
afford abutilin B (2) (22 mg).
20
([α]D = +102.1).
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