New secondary metabolites from Asphodelus tenuifolius

Article abstract of DOI:10.1002/hlca.201100261

Asphorins A and B (1 and 2, resp.), two new triterpene glycosides, have been isolated along with a new chromone, 3, from the AcOEt subfraction of the MeOH extract of the whole plant of Asphodelus tenuifolius. Their structures were elucidated by spectral analysis including 2D-NMR spectroscopic experiments. Copyright

Full text of DOI:10.1002/hlca.201100261

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Helvetica Chimica Acta – Vol. 95 (2012)
New Secondary Metabolites from Asphodelus tenuifolius
by Muhammad Safdera), Rashad Mehmooda), Bakhat Alia), Uzma Rasheed Mughala),
Abdul Malik*a), and Abdul Jabbarb)
a) International Center for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry,
University of Karachi, Karachi-75270, Pakistan
(phone: þ 92-21-4824926; fax: þ 92-21-4819018-9; e-mail: abdul.malik@iccs.edu)
b) Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan
Asphorins A and B (1 and 2, resp.), two new triterpene glycosides, have been isolated along with a
new chromone, 3, from the AcOEt subfraction of the MeOH extract of the whole plant of Asphodelus
tenuifolius. Their structures were elucidated by spectral analysis including 2D-NMR spectroscopic
experiments.
Introduction. – Asphodelaceae is one of the subfamilies of Liliaceae, and comprises
15 genera and 780 species, which are distributed in tropical, subtropical, and temperate
regions around the world, but mainly in Southern Africa. Among these, three genera
and eleven species are native to Pakistan. One of the genera is Asphodelus, which
comprises 20 species distributed from Mediterranean to western Asia. One of its
species is Asphodelus tenuifolius Cav. (syn. Asphodelus fistulosus var.), which is found
in northern Africa, and southwest regions of Asia and Europe. It commonly grows in
the Cholistan desert of Pakistan. This plant is used by local population as a diuretic [1].
Literature survey revealed that only one triterpene has so far been reported from this
plant [2]. The chemotaxonomic and ethnopharmacological importance of the genus
Asphodelus prompted us to carry out further phytochemical studies on A. tenuifolius.
As a result, we have isolated two new triterpenoid glycosides, named asphorins A and B
(1 and 2, resp.), along with a new chromone (3).
Results and Discussion. – The MeOH extract of the whole plant of A. tenuifolius
Cav. was suspended in H2O and successively extracted with hexane, CHCl3, AcOEt,
and BuOH. The AcOEt-soluble subfraction was subjected to column chromatographic
techniques (cf. the Exper. Part) to obtain compounds 1 – 3, respectively.
Asphorin A (1) was obtained as colorless crystals and gave positive Lieber-
mannꢀBurchard test for a triterpene and brisk effervescence with diluted NaHCO3,
indicating the presence of free carboxylic acid moiety. The HR-FAB-MS (negative-ion
mode) of 1 showed a quasi-molecular-ion ([M ꢀ H]ꢀ) peak at m/z 809.4324, consistent
with the molecular formula C42H65O15. The EI-MS exhibited a prominent peak at m/z
604 ([M ꢀ CO2 ꢀ 162]þ). Its intensity allowed us to locate the hexose moiety at C(17).
The loss of another hexose unit led to another fragment-ion peak at m/z 442. The peaks
arising from the retro-DielsꢀAlder fragmentation at m/z 440 and 370 indicated the
presence of a hexose moiety in rings A/B and two carboxylic moieties along with a
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 95 (2012)
145
hexose unit in rings C/D. The IR spectrum evidenced the presence of a OH group
(3425 cmꢀ1), ester and carboxylic acid CO functionalities (1732 cmꢀ1), an olefinic bond
(1638 cmꢀ1), and CꢀO moiety (1103 cmꢀ1). The UV spectrum exhibited absorption
maxima at 268 and 202 nm. The 13C-NMR (H-BB decoupled) and DEPT spectra
showed 42 well-resolved signals corresponding to six Me, eleven CH2, 17 CH groups,
and eight quaternary C-atoms (Table 1). The most downfield signals at d(C) 178.0 and
176.5 were assigned to ester and carboxylic acid moieties, respectively. The olefinic C-
atoms resonated at d(C) 133.2 and 129.5, while the O-bearing CH C-atom signal
appeared at d(C) 88.7. The six Me groups resonated in the range of d(C) 28.0 – 16.6. The
signals at d(C) 106.9 and 95.7 were assigned to two anomeric C-atoms of the hexose
moieties, while the signals of their O-bearing CH2 and CH C-atoms were observed in
1
the range of d(C) 79.3 – 62.3. In the H-NMR spectrum, the signals of the olefinic H-
atom of a trisubstituted C¼C bond and an O-bearing CH H-atom were observed at
d(H) 5.97 (br. s) and 3.19 (dd, J ¼ 11.3, 4.5), respectively. The signals of tertiary Me
groups appeared as singlets at d(H) 1.21, 1.02, 0.93, and 0.87, and of two secondary Me
groups as doublets at d(H) 1.17 (d, J ¼ 6.4) and 0.73 (d, J ¼ 6.6). The anomeric H-atoms
gave rise also to doublets at d(H) 6.37 (d, J ¼ 7.8) and 4.78 (d, J ¼ 5.3). The O-bearing
CH H-atoms of the sugar moieties resonated in the range of d(H) 4.40 – 3.93, while the
signals of the O-bearing CH2 H-atoms were observed in the range of d(H) 4.59 – 4.27.
The above data corresponded to an ursene-type triterpene with two of the Me groups
oxidized to carboxylic functions. The NMR data exhibited close resemblance to those
previously reported for quinovic acid [3] except the downfield shifts of both HꢀC(3)
and C(3) signals, and key HMBCs (Fig. 1), evidencing the presence of hexose moieties
Fig. 1. Structures and key HMBCs of asphorins A and B (1 and 2, resp.)

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Helvetica Chimica Acta – Vol. 95 (2012)
at C(3) and C(28). The acid hydrolysis of compound 1 afforded a binary mixture of
glycones, which could be identified as those of d-mannose and d-glucose by TLC as
well as by comparison of retention times of their tetramethylsilane (TMS) ethers with
corresponding standards in gas chromatography (GC). The large values of the coupling
constant allowed us to assign b-configuration to both the hexose units. The aglycone
could be identified as quinovic acid by comparison of its physical and spectral data with
those reported in literature [3]. Thus, asphorin A (1) was assigned the structure (3b)-3-
(b-d-mannopyranosyloxy)urs-12-ene-27,28-dioic acid 28-O-b-d-glucopyranosyl ester
(Fig. 1).
Asphorin B (2) was obtained as colorless crystals which gave similar tests as those of
1. The HR-FAB-MS (positive-ion mode) of 2 showed a quasi-molecular-ion ([M þ
H]þ) peak at m/z 729.3525, which corresponded to the molecular formula
C36H57O13S. The EI-MS exhibited prominent peaks at m/z 486 ([M ꢀ SO3hexose]þ)
and 468 ([M ꢀ SO3hexose ꢀ H2O]þ). The retro-DielsꢀAlder fragment-ion peaks at m/z
450 and 278 indicated the presence of a hexose moiety in rings A/B and two carboxy
functionalities in rings C/D. The presence of sulfohexose moiety was further confirmed
by fragment ions at m/z 97 and 80, respectively. The IR and UV spectra were similar to
those of 1. The 13C-NMR (H-BB decoupled and DEPT) spectra showed 36 well-
resolved signals corresponding to six CH3, ten CH2, twelve CH groups, and eight
quaternary C-atoms (Table 1). The carboxy functions gave rise to downfield signals at
d(C) 177.8 and 176.8. The olefinic C-atom signals appeared at d(C) 134.0 and 128.8,
while those of the O-bearing CH group were at d(C) 89.8. The six Me groups resonated
in the range of d(C) 28.1 – 16.5. The spectrum further exhibited the signals of the
anomeric C-atom of a hexose moiety at d(C) 104.1. The signal of the O-bearing CH2
and O-bearing CH group C-atoms of the sugar moiety were observed in the range of
d(C) 81.0 – 62.7.
1
The H-NMR spectrum showed signals of the olefinic H-atom and the O-bearing
CH H-atom of the aglycone moiety at d(H) 5.96 (br. s) and 3.12 (dd, J ¼ 11.1, 4.2),
respectively. The tertiary Me groups were attributed to singlets at d(H) 1.25, 1.11, 1.02,
and 0.78, while the two secondary Me groups resonated as doublets at d(H) 1.19 (d, J ¼
6.0) and 0.77 (d, J ¼ 6.4). The spectrum also showed the doublet for an anomeric H-
atom at d(H) 4.80 (d, J ¼ 5.1). The other O-bearing CH and O-bearing CH2 H-atoms of
hexose moiety resonated in the range of d(H) 4.37 – 3.83 and 4.45 – 4.26, respectively.
The NMR data exhibited a close resemblance to those reported for 3-(2-O-sulfo-b-d-
glucopyranosyl)quinovic acid [4], except slight differences in the chemical shifts of the
hexose unit. Acid hydrolysis of compound 2 gave quinovic acid and a glycone, the latter
was identified as d-mannose by comparison of retention time of its TMS ether with that
of a standard in GC. The large coupling constant of the anomeric H-atom allowed us to
assign b-d-configuration to the sugar unit. One of the O-bearing CH H-atom of d-
mannose resonated comparatively at d(H) 5.00 (dd, J ¼ 5.2, 5.1) due to the presence of
a sulfate moiety and could subsequently be assigned to C(2’) (d(C) 81.0) through
1
HMBCs (Fig. 1). Comparison of H- and 13C-NMR data of compound 2 with that of
quinovic acid revealed the downfield shifts of both C(3) and HꢀC(3), allowing us to
assign the b-d-mannoside moiety to this position. On the basis of these evidences, the
structure of asphorin B (2) could be assigned as (3b)-3-(2-O-sulfo-b-d-mannopyrano-
syloxy)urs-12-ene-27,28-dioic acid (Fig. 1).

Helvetica Chimica Acta – Vol. 95 (2012)
147
Table 1. 1H- and 13C-NMR Data (at 500 and 125 MHz, resp.), and Key HMBCs of Compounds 1 and 2.
Recorded in C5D5N; d in ppm, J in Hz.
1
2
d(H)
d(C) HMBC
d(H)
d(C) HMBC
CH2(1)
CH2(2)
HꢀC(3)
1.38 – 1.40 (m),
0.89 – 0.92 (m)
2.04 – 2.06 (m),
1.66 – 1.69 (m)
3.19 (dd,
39.0
26.7
–
–
1.41 – 1.44 (m),
0.92 – 0.94 (m)
2.06 – 2.09 (m),
1.87 – 1.89 (m)
38.9
26.4
–
–
88.7 2, 4, 5, 1’
3.12 (dd, J ¼ 11.1, 4.2)
89.8 2, 4, 5, 1’
J ¼ 11.3, 4.5)
C(4)
–
39.4
–
–
39.5
–
HꢀC(5)
0.86 (d, J ¼ 10.6)
1.50 – 1.53 (m),
1.30 – 1.33 (m)
1.88 – 1.92 (m),
1.68 – 1.73 (m)
–
55.7 3, 4, 10
0.82 (d, J ¼ 12.0)
55.7 3, 4, 10
CH2(6)
18.5
37.5
40.1
–
–
–
1.37 – 1.41 (m)
18.5
37.5
40.0
–
–
–
CH2(7)
1.79 – 1.85 (m),
1.64 – 1.66 (m)
–
C(8)
HꢀC(9)
2.49 – 2.53 (m)
–
2.06 – 2.11 (m),
1.90 – 1.94 (m)
47.2 8, 10, 11, 12
2.57 – 2.60 (m)
–
2.04 – 2.08 (m),
1.87 – 1.90 (m)
5.96 (br. s)
–
47.1 8, 10, 11, 12
C(10)
36.9
23.3
–
–
36.9
23.3
–
–
CH2(11)
HꢀC(12) 5.97 (br. s)
129.5 11, 13, 14
128.8 11, 13, 14
C(13)
–
–
133.2
56.7
–
–
134.0
56.8
–
–
C(14)
–
CH2(15)
2.60 – 2.64 (m),
2.50 – 2.54 (m)
2.25 – 2.30 (m),
2.00 – 2.04 (m)
–
25.5 13, 14, 17, 27
2.44 – 2.49 (m),
2.24 – 2.26 (m)
2.18 – 2.22 (m),
1.76 – 1.78 (m)
–
25.5 13, 14, 17, 27
CH2(16)
C(17)
26.1
48.9
–
–
26.5
48.8
–
–
HꢀC(18) 2.68 (d, J ¼ 11.3)
54.6 12, 13, 14, 17, 2.78 (d, J ¼ 11.4)
55.0 12, 13, 14,
17, 19, 28, 29
19, 28, 29
HꢀC(19) 1.46 – 1.49 (m)
HꢀC(20) 0.87 – 0.91 (m)
37.5
39.0
30.2
–
–
–
1.36 – 1.40 (m)
0.77 – 0.80 (m)
1.36 – 1.39 (m),
1.30 – 1.34 (m)
1.93 – 1.96 (m)
37.7
39.4
30.6
–
–
–
CH2(21)
1.39 – 1.42 (m),
1.26 – 1.30 (m)
1.98 – 2.02 (m),
1.77 – 1.80 (m)
1.21 (s)
CH2(22)
36.4
–
37.1
–
Me(23)
Me(24)
Me(25)
Me(26)
C(27)
28.0 3, 4, 5, 24
17.0 3, 4, 5, 23
16.6 1, 9, 10
19.2 7, 8, 9, 14
176.5
178.0
1.25 (s)
1.11 (s)
0.78 (s)
1.02 (s)
–
–
28.1 3, 4, 5, 24
17.1 3, 4, 5, 23
16.5 1, 9, 10
18.9 7, 8, 9, 14
176.8
177.8
0.93 (s)
0.87 (s)
1.02 (s)
–
–
–
–
–
C(28)
–
Me(29)
Me(30)
HꢀC(1’)
HꢀC(2’)
HꢀC(3’)
HꢀC(4’)
HꢀC(5’)
1.17 (d, J ¼ 6.4)
0.73 (d, J ¼ 6.6)
4.78 (d, J ¼ 5.3)
3.96 – 3.99 (m)
4.12 – 4.16 (m)
4.18 – 4.22 (m)
3.93 – 3.95 (m)
18.1 18, 19, 20
21.2 19, 20, 21
106.9 3, 2’, 3’
75.7 1’, 3’, 4’
1.19 (d, J ¼ 6.0)
0.77 (d, J ¼ 6.4)
4.80 (d, J ¼ 5.1)
5.00 (dd, J ¼ 5.4, 5.1)
4.37 – 4.41 (m)
4.14 (t, J ¼ 9.0)
3.83 – 3.88 (m)
18.3 18, 19, 20
21.3 19, 20, 21
104.1 3, 2’, 3’
81.0 1’, 3’, 4’
78.9
71.8
79.3
–
–
–
78.2
71.6
77.7
–
–
–

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Helvetica Chimica Acta – Vol. 95 (2012)
Table 1 (cont.)
1
2
d(H)
d(C)
HMBC
–
d(H)
d(C)
HMBC
–
CH2(6’)
4.44 – 4.47 (m),
4.37 – 4.40 (m)
6.37 (d, J ¼ 7.8)
4.30 – 4.33 (m)
4.20 – 4.24 (m)
4.33 – 4.37 (m)
4.38 – 4.40 (m)
4.55 – 5.59 (m),
4.27 – 4.30 (m)
63.0
4.43 – 4.47 (m),
62.7
4.26 (dd, J ¼ 5.1, 5.1)
HꢀC(1’’)
HꢀC(2’’)
HꢀC(3’’)
HꢀC(4’’)
HꢀC(5’’)
CH2(6’’)
95.7
74.1
78.2
71.2
78.7
62.3
28
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Compound 3 (Fig. 2) was obtained as an amorphous powder, which gave purple
coloration with FeCl3, typical for a 5-hydroxychromone [5]. The HR-EI-MS showed
the Mþ peak at m/z 626.5269, which corresponded to the molecular formula C41H70O4.
The IR spectrum indicated the presence of OH group (3411 cmꢀ1), conjugated CO
functionality (1671 cmꢀ1), conjugated C¼C bond (1630 cmꢀ1), and an aromatic moiety
(1543, 1503 cmꢀ1). The UV spectrum showed absorption maxima at 336, 314, 290, 248,
and 216, which were characteristic of a chromone [6]. The 13C-NMR (H-BB decoupled
and DEPT) spectra (Table 2) showed the signal of the CO C-atom at d(C) 182.7. The
O-bearing aromatic C-atoms exhibited signals at d(C) 170.7, 162.2, 160.0, and 158.6.
The aromatic quaternary C-atoms gave signals at d(C) 108.8 and 106.1, and aromatic
CH C-atoms resonated at d(C) 107.9 and 93.2. A CH2 group showed signal at d(C) 34.2
indicating its association with the conjugated system, and the other CH2 groups of the
long chain resonated in the region of d(C) 31.9 – 22.7. An aromatic Me group resonated
at d(C) 7.0. The 1H-NMR spectrum (Table 2) showed a downfield signal at d(H) 13.01
for a chelated OH group at C(5). The spectrum further displayed two singlets at d(H)
6.31 (s, 1 H) and 5.99 (s, 1 H), which were assigned to C(6) and C(3), respectively, on
the basis of HMBCs. The Me group resonated at d(H) 2.11 (s, 3 H) suggesting its
attachment to the aromatic ring. The upfield region of the spectrum evidenced the
presence of a long hydrocarbon chain with one of the CH2 H-atoms resonating as a
triplet at d(H) 2.53 (t, J ¼ 7.5). It showed COSY correlations with neighboring CH2 H-
atoms at d(H) 1.61 – 1.64. Further 56 CH2 H-atom signals were observed as a broad
hump in the range of d(H) 1.28 – 1.20. The terminal Me H-atoms gave a triplet at d(H)
0.84 (t, J ¼ 6.8). In the HMBC experiment, the HꢀC(6) signal at d(H) 6.31 showed 2J
3
correlations with those of C(7) (d(C) 162.2) and C(5) (d(C) 158.6), as well as J
correlations with those of C(8) (d(C) 106.1) and C(4a) (d(C) 108.8). The HꢀC(3)
2
signal at d(H) 5.99 showed J correlations with those of C(4) (d(C) 182.7) and C(2)
(d(C) 170.7) as well as 3J correlations with those of C(4a) (d(C) 108.8) and C(1’) (d(C)
2
34.2). The aromatic Me signal at d(H) 2.11 showed J correlation with those of C(8)
3
(d(C) 106.1), and J correlations with those of C(7) (d(C) 162.2) and C(8a) (d(C)
160.0). The remaining HMBCs are compiled in Table 2. In the EI-MS spectrum, the
peaks at m/z 219 and 206 [7] appeared due to the losses of nonacosanyl and triacontanyl
moieties, respectively. The diagnostic peak at m/z 167, resulting from retro-DielsꢀAlder

Helvetica Chimica Acta – Vol. 95 (2012)
149
fragmentation, confirmed the presence of the Me group in ring A. The spectrum also
exhibited fragment-ion peaks, differing from each other by 14 a.m.u., of the hydro-
carbon chain. On the basis of the spectral data, the structure of compound 3 was
assigned as 2-hentriacontyl-5,7-dihydroxy-8-methyl-4H-1-benzopyran-4-one (Fig. 2).
Fig. 2. Structure of compound 3
Table 2. 1H- and 13C-NMR Data (at 500 and 125 MHz, resp.), and HMBCs of Compound 3. Recorded in
CD3OD; d in ppm, J in Hz.
d(H)
d(C)
HMBC
C(2)
–
170.7
107.9
182.7
108.8
158.6
93.2
162.2
106.1
160.0
34.2
26.8
29.7 – 28.9
31.9
22.7
14.1
–
HꢀC(3)
5.99 (s)
–
–
–
6.31 (s)
–
–
–
2.53 (t, J ¼ 7.5)
1.61 – 1.64 (m)
1.28 – 1.20 (m)
1.28 – 1.20 (m)
1.28 – 1.20 (m)
0.84 (t, J ¼ 6.8)
2.11 (s)
1’, 2, 4, 4a
–
–
–
5, 7, 8, 4a
–
–
–
C(4)
C(4a)
C(5)
HꢀC(6)
C(7)
C(8)
C(8a)
CH2(1’)
CH2(2’)
CH2(3’ – 28’)
CH2(29’)
CH2(30’)
Me(31’)
Me(1’’)
2, 3, 2’, 3’
2, 1’, 3’
–
–
29’, 31’
29’, 30’
7, 8, 8a
7.0
The authors are grateful to Higher Education Commission of Pakistan for financial grant to M. S.
Experimental Part
General. Column chromatography (CC): silica gel (SiO2; 230 – 400 mesh; E. Merck, D-Darmstadt).
TLC: silica gel 60-F254 plates (E. Merck, D-Darmstadt). GC: Schimadzu gas chromatograph (GC-9A);
3% OV-1 silanized Chromosorb W column; column temp., 1808; injection port and detector temp., 275 –
3008; flow rate, 35 ml/min; flame-ionization detector. M.p.: Gallenkemp apparatus. Optical rotations:
JASCO P-2000 polarimeter. UV Spectra: Hitachi UV-3200 spectrophotometer; lmax (log e) in nm. IR
Spectra: JASCO 302-A spectrometer; in KBr; ˜n in cmꢀ1. NMR Spectra: Bruker AMX-500 instrument.
EI-MS: Finnigan MAT 312 mass spectrometer. HR-FAB-MS: JEOL JMS-HX-110 mass spectrometer,
glycerol as matrix.
Plant Material. The whole-plant material of Asphodelus tenuifolius Cav. (Asphodelaceae; 8 kg) was
collected from Cholistan Desert (Bahawalpur) in 2006 and identified by Dr. Muhammad Arshad, Plant
Taxonomist, Cholistan Institute of Desert Studies (CIDS), The Islamia University of Bahawalpur,
Bahawalpur, where a voucher specimen has been deposited (Voucher specimen No. 28/CIDS/06).

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Helvetica Chimica Acta – Vol. 95 (2012)
Extraction and Isolation. The shade-dried whole-plant material (8 kg) was cut into small pieces and
extracted with MeOH (3 ꢁ 30 l, 10 d each) at r.t. The combined extract was evaporated under reduced
pressure to yield a residue (300 g), which was suspended in H2O (1.0 l), and partitioned with hexane
(45 g), CHCl3 (85 g), AcOEt (75 g), and BuOH (45 g). The AcOEt-soluble subfraction (75 g) was
subjected to CC (hexane/CHCl3, CHCl3, and CHCl3/MeOH in increasing order of polarity) to yield ten
fractions, Frs. A – J. The Fr. B (3 g), obtained with hexane/CHCl3 2 :8, was re-chromatographed and
eluted with same solvent system to yield a major compound with traces of impurities. It was purified by
prep. TLC (hexane/CHCl3 0.5 :9.5) to afford compound 3 (20 mg). Fr. F (4 g), which was obtained with
CHCl3/MeOH 9 :1, was chromatographed (CHCl3/MeOH 9.2 :0.8) to give a semi-pure compound, which
was further chromatographed (CHCl3/MeOH 9 :1) to provide compound 1 (18 mg). The Fr. H (3.5 g),
obtained with CHCl3/MeOH 8 :2, was triturated with acetone, and the acetone-insoluble residue was
chromatographed (CHCl3/MeOH 8.2 :1.8) to yield a semi-pure compound, which was purified by prep.
TLC (CHCl3/MeOH 7.5 :2.5) to give compound 2 (15 mg).
Asphorin A (¼1-O-[(3b)-27-Hydroxy-3-(b-d-mannopyranosyloxy)-27,28-dioxours-12-en-28-yl]-b-
d-glucopyranose; 1). Colorless crystals. M.p. 194 – 1958. [a]2D5 ¼ þ15 (c ¼ 0.065, MeOH). UV (MeOH):
268 (3.2), 202 (1.9). IR (KBr): 3425, 1732, 1638, 1103. 1H- and 13C-NMR: see Table 1. FAB-MS (neg.):
809 ([M ꢀ H]ꢀ), 603 ([M ꢀ H ꢀ hex ꢀ CO2]ꢀ). EI-MS: 604 (4, [M ꢀ hex ꢀ CO2]þ), 590 (4, [M ꢀ hex ꢀ
CO2 ꢀ Me]þ), 440 (5), 425 (10), 409 (15), 370 (10), 287 (16), 261 (11), 247 (11), 207 (14), 203 (15), 189
(19), 135 (40), 95 (54), 81 (45), 73 (66), 69 (100), 55 (99). HR-FAB-MS (neg.): 809.4320 ([M ꢀ H]ꢀ,
C42H65Oꢀ15 ; calc. 809.4324).
Asphorin B (¼(3b)-3-[(2-O-Sulfo-b-d-mannopyranosyl)oxy]urs-12-ene-27,28-dioic Acid; 2). Color-
less crystals. M.p. 270 – 2718. [a]2D5 ¼ þ26 (c ¼ 0.09, MeOH). UV (MeOH): 270 (4.2), 204 (1.3). IR (KBr):
3418, 1729, 1634. 1H- and 13C-NMR: see Table 1. EI-MS: 486 ([M ꢀ SO3hexose]þ), 468 (4, [M ꢀ
SO3hexose ꢀ H2O]þ), 450 (12), 442 (20), 427 (42), 409 (46), 287 (39), 278 (18), 273 (33), 261 (30),
247 (15), 207 (23), 203 (27), 189 (50), 135 (83), 97 (30), 95 (87), 83 (100), 80 (60), 69 (80), 55 (92). HR-
FAB-MS (pos.): 729.3525 ([M þ H]þ, C36H57O13Sþ; calc. 729.3520).
Compound 3 (¼2-Hentriacontyl-5,7-dihydroxy-8-methyl-4H-1-benzopyran-4-one). Amorphous
powder. M.p. 153 – 1558. UV (MeOH): 366 (4.1), 314 (4.3), 290 (3.8), 248 (3.9), 216 (2.4). IR (KBr):
1
3411, 1671 (CO), 1630, 1543, 1500. H- and 13C-NMR: see Table 2. EI-MS: 626 (5, Mþ), 598 (73), 586
(12), 570 (52), 541 (11), 513 (11), 499 (10), 373 (13), 219 (100), 206 (36), 167 (20), 120 (28), 57 (13), 43
(16). HR-EI-MS: 626.5269 (Mþ, C41H70Oþ4 ; calc. 626.5274).
Acid Hydrolysis of 1 and 2. The MeOH soln. of each compound (5 mg) was hydrolyzed with 2n aq.
CF3COOH (8 ml) was refluxed for 4 h at 908 and extracted with CH2Cl2 (3 ꢁ 10 ml). Evaporation of the
combined org. phase provided a pure crystalline compound (m.p. 297 (dec.) and [a]2D5 ¼ þ87). It could be
identified as quinovic acid by comparison of physical and spectral data with those reported in [3]. The aq.
layer was repeatedly concentrated under reduced pressure with MeOH until neutral, and then analyzed
by TLC (SiO2; CHCl3/MeOH/H2O 8 :5 :1) by comparison with authentic sample. The sugar residue was
dissolved in dry pyridine (1.0 ml) and stirred with l-cysteine methyl ester hydrochloride (1.5 mg) at 608
for 70 min. After concentration, 1-(trimethylsilyl)-1H-imidazole was added, and the mixture was stirred
at 608 for 25 min. The supernatant was concentrated under N2 and analyzed by GC. d-Glucose and d-
mannose were detected from 1 by co-injection of the hydrolysate with standard silylated samples with tR
values of 14.1 and 13.2 min, resp. In case of 2, only d-mannose was detected with a tR value of 13.1 min
[8].
REFERENCES
[1] S. I. Ali, ꢂFlora of Pakistanꢃ, Missouri Botanical Press, Missouri, 2004, Vol. 211, p. 1.
[2] M. Safder, M. Imran, R. Mehmood, A. Malik, N. Afza, L. Iqbal, M. Latif, J. Asian Nat. Prod. Res.
2009, 11, 945.
[3] K. Pçllmann, S. Gagel, M. H. A. Elgamal, K. H. Shaker, K. Seifert, Phytochemistry 1997, 44, 485.
[4] D. Smati, A.-C. Mittaine-Offer, T. Miyamoto, V. Hammiche, M.-A. Lacaille-Dubois, Helv. Chim.
Acta 2007, 90, 712.

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[5] F. M. Dean, ꢂNaturally Occurring Oxygen Ring Compoundꢃ, Butterworths, London, 1963.
[6] G. N. Belofsky, P. R. Jensen, M. K. Renner, W. Fenical, Tetrahedron 1998, 54, 1715.
[7] R. G. Cooke, J. G. Down, Tetrahedron Lett. 1970, 13, 1039.
[8] A. Itoh, T. Tanahashi, N. Nagakura, T. Nishi, Chem. Pharm. Bull. 2003, 51, 1335.
Received June 20, 2011