Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis

Article abstract of DOI:10.1080/10286020.2012.680440

Four new sphingolipids: nudicaulin A [(2S,3S,4R,14E)-2-{[octadecanoyl] amino}tetraeicos-14-ene-1,3,4-triol; 1], nudicaulin B [(2S,3S,4R,14E)-2-{[(2R)- 2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 2], nudicaulin C [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3, 4-triol-1-O-β-d-glucopyranoside; 3], and nudicaulin D [(2S,3S,4R)-2-{[(2R, 3S,12E)-2,3-dihydroxyeicos-12-enoyl]amino}octadecane-1,3,4-triol; 4] together with 1-hexatriacontanol,-sitosterol, octadecyl 4-hydroxycinnamate, elaidic acid, cholesta-5,22-diene-3,7-diol, oleanolic acid, apigenin, and-sitosterol 3-O-β-d-glucopyranoside were isolated from the methanolic extract of the whole plant of Launaea nudicaulis. Their structures were elucidated using ¹H and ¹³C NMR spectra and 2D NMR analyses (HMQC, HMBC, and COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-FAB-MS) experiments and comparison with literature data of related compounds. Compounds 1-4 displayed moderate inhibitory potential against enzyme lipoxygenase in concentration-dependent manner with IC₅₀ value ranges 103-193M.

Full text of DOI:10.1080/10286020.2012.680440

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Lipoxygenase inhibitory sphingolipids
from Launaea nudicaulis
Naheed Riaz a , Shehla Parveen a , Muhammad Saleem a ,
Muhammad Shaiq Ali b , Abdul Malik b , Muhammad Ashraf c ,
Iftikhar Afzal c & Abdul Jabbar a
a Department of Chemistry , Baghdad-ul-Jadeed Campus, The
Islamia University of Bahawalpur , 63100 , Bahawalpur , Pakistan
b HEJ Research Institute of Chemistry, International Centre for
Chemical and Biological Sciences (ICCBS), University of Karachi ,
Karachi , 75270 , Pakistan
c Department of Biochemistry and Biotechnology , Baghdad-
ul-Jadeed Campus, The Islamia University of Bahawalpur ,
Bahawalpur , 63100 , Pakistan
Published online: 15 May 2012.
To cite this article: Naheed Riaz , Shehla Parveen , Muhammad Saleem , Muhammad Shaiq Ali ,
Abdul Malik , Muhammad Ashraf , Iftikhar Afzal & Abdul Jabbar (2012) Lipoxygenase inhibitory
sphingolipids from Launaea nudicaulis , Journal of Asian Natural Products Research, 14:6, 545-554,
DOI: 10.1080/10286020.2012.680440
To link to this article: http://dx.doi.org/10.1080/10286020.2012.680440
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Journal of Asian Natural Products Research
Vol. 14, No. 6, June 2012, 545–554
Lipoxygenase inhibitory sphingolipids from Launaea nudicaulis
Naheed Riaza, Shehla Parveena, Muhammad Saleema, Muhammad Shaiq Alib,
Abdul Malikb, Muhammad Ashrafc, Iftikhar Afzalc and Abdul Jabbara*
aDepartment of Chemistry, Baghdad-ul-Jadeed Campus, The Islamia University of Bahawalpur,
63100 Bahawalpur, Pakistan; HEJ Research Institute of Chemistry, International Centre for
b
Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi 75270, Pakistan;
cDepartment of Biochemistry and Biotechnology, Baghdad-ul-Jadeed Campus, The Islamia
University of Bahawalpur, Bahawalpur 63100, Pakistan
(Received 26 October 2011; final version received 26 March 2012)
This paper is dedicated to Dr Muhammad Arshad (late), Ex-Director CIDS, The Islamia University
of Bahawalpur, whose death (March 31, 2011) is a big loss for the Institute as well as for the
University
Four new sphingolipids: nudicaulin A [(2S,3S,4R,14E)-2-{[octadecanoyl]amino}
tetraeicos-14-ene-1,3,4-triol; 1], nudicaulin B [(2S,3S,4R,14E)-2-{[(2R)-2-hydroxy-
octadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol; 2], nudicaulin C [(2S,3S,4R,
14E)-2-{[(2R)-2-hydroxyoctadecanoyl]amino}tetraeicos-14-ene-1,3,4-triol-1-O-b-D-
glucopyranoside; 3], and nudicaulin D [(2S,3S,4R)-2-{[(2R,3S,12E)-2,3-dihydroxy-
eicos-12-enoyl]amino}octadecane-1,3,4-triol; 4] together with 1-hexatriacontanol,
b-sitosterol, octadecyl 4-hydroxycinnamate, elaidic acid, cholesta-5,22-diene-3,7-diol,
oleanolic acid, apigenin, and b-sitosterol 3-O-b-D-glucopyranoside were isolated from
the methanolic extract of the whole plant of Launaea nudicaulis. Their structures were
elucidated using 1H and 13C NMR spectra and 2D NMR analyses (HMQC, HMBC, and
COSY) in combination with mass spectrometry (EI-MS, HR-EI-MS, FAB-MS, and HR-
FAB-MS) experiments and comparison with literature data of related compounds.
Compounds 1–4 displayed moderate inhibitory potential against enzyme lipoxygenase
in concentration-dependent manner with IC50 value ranges 103–193 mM.
Keywords: Launaea nudicaulis; sphingolipids; secondary metabolite; lipoxygenase
inhibition
1. Introduction
sesquiterpene lactones, triterpenes, steroids,
flavonoids, and coumarins from Launaea
species [7–12]. Launaea nudicaulis is an
important member of this genus, wildly
growing in Cholistan Desert and is known to
be effective against itches, ulcers, cuts,
swellings, bilious fever, eczema eruptions,
cancer, microbial pathogens, insects, and
rheumatism [13,14]. The leaves of L.
nudicaulis are used as antipyretic in
children, whereas its latex is used to relieve
The genus Launaea of Compositae family is
known for antitumor, insecticidal, and
cytotoxic activities and used in folk medicine
against stomach ache and skin diseases [1–
3]. In Pakistan, this genus is represented by
20 species mostly with galactagogue,
soporific, diuretic, and aperient properties
[4]. Some species are important for the
treatment of infected wounds and fever [5,6].
Literature survey reveals the presence of
*Corresponding author. Email: abdul_jabbar06chem@yahoo.com; nrch322@yahoo.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.680440
http://www.tandfonline.com

546
N. Riaz et al.
constipation [13]. The diverse biological
activities of L. nudicaulis prompted us to
carryoutphytochemicalstudiesonthisplant.
Herein, we report the isolation, structure
elucidation, and biological studies of four
new sphingolipids (1–4) together with
known metabolites: 1-hexatriacontanol, b-
sitosterol, octadecyl 4-hydroxycinnamate,
elaidic acid cholesta-5,22-diene-3,7-diol,
oleanolic acid, apigenin, and b-sitosterol 3-
Ob-D-glucopyranoside from the n-hexane-
soluble fraction of methanolic extract of the
whole plant material.
secondary amide (1650cm21), and olefinic
(1625 cm21) groups. The molecular formula
C42H83NO4 was deduced by HR-EI-MS
showing molecular ion peak at m/z 665.6330
[M]þ. It indicated that the molecule has two
1
double bond equivalences. The H NMR
spectrum of 1 (Table 1) displayed signal for
a secondary amide at d 6.75 (1H, d,
J ¼ 8.0 Hz); an oxygenated methylene at d
3.94(1H, dd, J ¼ 11.5, 3.5 Hz) and 3.57 (1H,
dd, J ¼ 11.5, 5.5Hz); two oxymethines at d
3.47 (1H, dt, J ¼ 5.3, 4.1 Hz) and 3.43 (1H,
dd, J ¼ 4.5, 4.1 Hz). A signal at d 3.97 (1H,
m) was ascribed for the methine proton
vicinal to the nitrogen atom of the amide
group. The 1HNMRspectrumalsodisplayed
signals for a double bond and two primary
methyls. The 13C NMR spectrum of 1
(Table 1) disclosed the signal for an amide
carbonyl (d 174.4), a methine carbon
(d 51.6), one double bond (d 130.8, 129.0),
oxygenated methines and a methylene (d
75.7, 72.4, 60.9), and aliphatic chain (d
32.6–29.2). These data fully supported the
1H NMR data and declared 1 to be a
sphingolipid [15,16]. A methylene triplet at d
2.10 (J ¼ 8.0 Hz) indicated the absence of
hydroxyl group at C-20 [15]. The entire
2. Results and discussion
The methanolic extract of L. nudicaulis
was divided into n-hexane, ethyl acetate,
and n-butanol fractions. The n-hexane
soluble fraction on column chromatog-
raphy (CC) yielded four new sphingolipids
(1–4), together with above-mentioned
known compounds.
Compound 1 was isolated as a colorless
amorphous powder. The UV spectrum of 1
indicated the absence of UV-active chromo-
phores, while infrared (IR) spectrum showed
the presence of hydroxyl (3640 cm21),
Table 1. 1H and 13C NMR spectral data, HMBC, and COSY correlations of 1 (CDCl3; 400,
100 MHz).
HMBC
(H ! C)
2,3
Position
1
dH (J in Hz)
dC
60.9
COSY (H ! H)
H-1/H-2
3.94 (1H, dd, 11.5, 3.5)
3.57 (1H, dd, 11.5, 5.5)
3.97 (1H, m,)
3.43 (1H, dd, 4.5, 4.1)
3.47 (1H, dt, 5.3, 4.1)
1.56, 1.32 (1H each, m)
1.15–1.30 (56H, br s)
2
3
4
5
51.6
75.7
72.4
32.6
1,3,4,10
1,2,4,5
2,3,5,6
3,4,6
H-2/H-1,3,N-H
H-3/H-2,4
H-4/H-3,5
H-5/H-4,6
6-12,17-23,
40-170
29.2–31.5
5,13,24,180
H-6,12/H-5,13;
H-17,23/H-16,24;
H-40,170/H-30,180
H-13,16/H-12,14,15,17
H-14,15/H-13,16
H-24,180/H-23,170
N-H/H-2
13,16
14,15
24,180
NH
10
20
30
1.86–1.93 (4H, m)
5.31 (2H, dt, 16.5, 5.2)
0.79 (6H, t, 7.0)
6.75 (1H, d, 8.0)
–
32.4
129.0, 130.8
13.9
14,15
13,16
23,170
1,2,3,10
–
10,30
20,40
–
174.4
36.4
25.7
–
2.10 (2H, t, 8.0)
1.51 (2H, m)
H-20/H-30
H-30/H-40

Journal of Asian Natural Products Research
547
sequence of the skeleton and the substi-
1
showed the molecular ion peak at m/z
681.6280 [M]þ corresponding to the
molecular formula C42H83NO5 indicating
that 2 could be an oxidative derivative of 1.
The 1H and 13C NMR spectra of 2 (Table 2)
were almost similar to those of 1 with a
minor difference of an additional signal for
oxymethine at d 3.94 (1H, dd, J ¼ 6.4,
3.6 Hz) correlated with the carbon at d 71.9.
The signal of CH2-20 observed in 1 was
missing in the NMR spectra of 2 further
supported that C-20 is hydroxylated
[16,19–22]. The positions of all the
substituents were assigned with the help
of 1H–1H COSY and HMBC spectra
(Table 2) and mass fragmentation patterns
(Figure 2). The length of the fatty acid and
the amine chains, and the position of double
bond were tentatively fixed by the combi-
nation of mass fragmentation pattern
(Figure 2) and methanolysis (see exper-
imental) as done for 1. The configuration at
the stereogenic centers was deduced by
optical rotations of 2 ([a]D ¼ þ39.7) and
its methanolysis products ([a ]D ¼ 27.1
and þ19.1), which were comparable with
those of sphingolipids with a 2S,3S,4R,20R
configurations [20–22]. Based on these
evidences, nudicaulin B (2) could be
assigned the structure (2S,3S,4R,14E)-2-
{[(2R)-2-hydroxyoctadecanoyl]amino}te-
traeicos-14-ene-1,3,4-triol.
tutions were fixed by H–1H COSY and
long-range HMBC correlations (Table 1).
The length of the fatty acid chain was
determined by characteristic fragments at
m/z 268, 369 and the amine chain containing
a double bond at m/z 427 and 341 (Figure 1).
Methanolysis [18] of 1 with methanolic HCl
provided the aliphatic amine and the methyl
ester of fatty acid, which after acetylation
[17] were analyzed by GC-MS and were
identified as methyl octadecanoate (m/z 298)
and 2-acetamino-1,3,4-triacetoxytetraeico-
sene (m/z 567). The position of double
bond was fixed between C-14 and C-15 by
permanganate/periodate oxidative cleavage
[18] of 2-acetamino-1,3,4-triacetoxytetrae-
cosene, yielding a mixture of carboxylic
acids which on methylation and GC-MS
analysis provided peaks for 2-acetamino-
1,3,4-triacetoxytetradecanoic acid (m/z 473)
and decanoic acid (m/z 186). The stereo-
chemistry at the three stereogenic centers
was determined by optical rotations of 1
([a]D ¼ þ16.9) and its methylated amine
([a]D ¼ þ11.2) which were found similar
with those having (2S,3S,4R)-configurations
[19–22]. Based on these evidences, 1 could
be assigned as (2S,3S,4R,14E)-2-{[octade-
canoyl]amino}tetraeicos-14-ene-1,3,4-triol
and named nudicaulin A.
Compound 2 was obtained as a white
amorphous powder. The IR spectrum of 2
was similar to that of 1. The HR-EI-MS
Compound 3 with a molecular formula
C49H94NO10, isolated as a colorless gummy
441
268
O
282
18'
CH3
1'
HN
2'
OH
24
CH3
14
HO
3
2
4
23
15
1
498
OH
386
552
384
356
339
Figure 1. Structure and mass fragmentation pattern of 1.

548
N. Riaz et al.
Table 2. 1H and 13C NMR spectral data, HMBC, and COSY correlations of 2 (CDCl3; 400,
100 MHz).
HMBC
(H ! C)
2,3
Position
1
dH (J in Hz)
dC
61.0
COSY (H ! H)
H-1/H-2
3.71 (1H, dd, 11.6, 4.0)
3.63 (1H, dd, 11.6, 4.8)
3.99 (1H, m)
3.45 (1H, dd, 4.2, 3.9)
3.41 (1H, dt, 6.3, 4.2)
1.59 (2H, m)
2
3
4
5
51.5
75.5
72.1
32.8
1,3,4,10
1,2,4,5
2,3,5,6
3,4,6
H-2/H-1,3
H-3/H-2,4
H-4/H-3,5
H-5/H-4,6
H-6,12/H-5,13;
H-17,23/
H-16,24; H-40,170/
H-30,180
6–12, 17–23,
40-170
1.15–1.29 (36H, br s)
28.5–31.3
5,13,24,180
13,16
14,15
24,180
NH
10
1.90 (4H, m)
32.5
130.8, 129.7
13.9
14,15
13,16
23,170
1,2,3,10,20
–
10,30,40
10,20,40
H-13,16/H-12,15,17
H-14,15/H-13,16
H-24,180/H-23,170
N-H/H-2
5.29 (2H, dt, 15.3, 6.8)
0.77 (6H, t, 6.4)
7.42 (1H, d, 8.8)
–
3.94 (1H, dd, 6.4, 3.6)
1.52 (2H, m)
–
175.7
71.9
32.3
–
20
H-20/H-30
30
H-30/H-20,40
solid, was found to be a spingolipid
glycoside based on its NMR data when
compared with those of 1 and 2. In addition
to the usual signals for a sphingolipid
skeleton, the 1H NMR spectrum of 3 (Table
3) displayed signals for a sugar moiety at d
4.15 (1H, d, J ¼ 7.6 Hz, H-100), 3.40 (1H, m,
H-200), 3.28 (1H, t, J ¼ 7.4 Hz, H-300), 3.23
(1H, t, J ¼ 7.4 Hz, H-400), 3.16 (1H, m, H-
500), 3.79 (1H, dd, J ¼ 10.8, 4.9 Hz, H-600),
and 3.61 (1H, dd, J ¼ 10.8, 2.9 Hz, H-600).
The 13C NMR spectrum of 3 (Table 3)
also supported the above data for a
glycosphingolid [21,22]. A downfield shift
(d 68.5) of C-1 was due to the sugar unit
attached to it, which was further confirmed
by HMBC correlation of anomeric methine
at d 4.15 with C-1 at d 68.5 (Table 3). In
addition to the presence of glucose moiety,
mass spectrometry (Figure 3), methanoly-
sis, and oxidative cleavage revealed that
compound3 has C17 fatty acid chain and C26
amine chain with a double bond at C-14,15
[20,23]. The stereochemistry at various
stereogenic centers was found similar to
those for 2. Based on these evidences,
456
283
O
OH
18'
CH3
1'
2'
HN
398
OH
24
CH3
14
HO
3
2
4
15
1
23
OH
113
383
279
167
339
Figure 2. Structure and mass fragmentation pattern of 2.

Journal of Asian Natural Products Research
549
Table 3. 1H and 13C NMR spectral data, HMBC, and COSY correlations of 3 (CDCl3; 400,
100 MHz).
HMBC
(H ! C)
Position
1
dH (J in Hz)
dC
68.5
COSY (H ! H)
H-1/H-2
3.92 (1H, dd, 11.3, 4.8)
3.71 (1H, dd, 11.3, 3.4)
4.09 (1H, m)
3.12 (1H, dd, 4.8, 3.8)
3.43 (1H, dt, 5.8, 4.8)
1.52 (2H, m)
2,3,100
2
3
4
51.5
76.1
74.2
32.1
1,3,4,10
1,2,4,5
2,3,5,6
3,4,6
H-2/H-1,3
H-3/H-2,4
H-4/H-3,5
H-5/H-4,6
H-6,12/H-5,13;
H-17,25/H-16,26;
H-40,160/H-30,170
H-13,16/H-12,14,15,17
H-14,15/H-13,16
H-26,170/H-25,160
N-H/H-2
5
6–12,
17–25,40-160
1.12–1.28 (58H, br s)
28.1–30.9
5,13,26,170
13,16
14,15
26,170
NH
10
1.86 (4H, m)
32.4
14,15
130.8, 129.7 13,16
5.26 (2H, dt, 17.3, 5.4)
0.77 (3H, t, 6.4)
7.48 (1H, d, 8.8)
–
3.88 (1H, t, 6.9)
1.64 (2H, m)
4.15 (1H, d, 7.6)
3.40 (1H, m)
3.28 (1H, t, 7.4)
3.23 (1H, t, 7.4)
3.16 (1H, m)
13.9
–
25,160
1,2,3,10
–
175.7
71.9
34.2
102.8
73.1
76.1
69.7
76.5
61.2
–
200
10,30
H-200/H-300
300
10,20,40
1,200,300
100,300,400
100,200,400,500
300,500
H-300/H-2 0,040
H-100/H-200
100
200
H-200/H-100,30000
H-3 /H-2 ,4
H-400/H-300,500
H-500/H-400,600
H-600/H-500
3
400
500
300,600
500,400
600
3.79 (1H, dd, 10. 8, 4.9)
3.61 (1H, dd, 10. 8, 2.9)
nudicaulin C (3) could be assigned the
structure (2S,3S,4R,14E)-2-{[(2R)-2-hydr-
oxyoctadecanoyl]amino}tetraeicos-14-ene-
1,3,4-triol-1-O-b-D-glucopyranoside.
The sphingolipid 4 was obtained as
colorless shiny powder with molecular
1
formula C38H75NO6. The H NMR spec-
corresponding to the carbon at d 72.8. This
information indicated that 4 could be an
oxidative derivative of 2 with slight
variation in lengths of both the chains
(Figure 4). All assignments were accom-
plished by careful analysis of mass frag-
mentation and 1D and 2D NMR data
(Table 4). The optical rotations of 4
([a]D ¼ þ29.9) and the subsequent metha-
nolysis products ([a]D ¼ þ9.6 and þ16.9)
trum of 4 (Table 4) was similar to that of 2
with an additional oxymethine signal at d
3.62 (1H, dt, J ¼ 6.2, 4.3 Hz, H-30),
269
O
17'
CH3
OH
1'
2'
HN
OH
OH
6''
HO 4''
HO
O
26
CH3
14
5''
2''
O
3
2
4
1
25
15
OH1''
3''
OH
307
141
662
394
694
367
Figure 3. Structure and mass fragmentation pattern of 3.

550
N. Riaz et al.
Table 4. 1H and 13C NMR spectral data, HMBC, and COSY correlations of 4 (CDCl3; 400,
100 MHz).
HMBC
(H ! C)
2,3
Position
1
dH (J in Hz)
dC
60.8
COSY (H ! H)
H-1/H-2
3.60 (1H,dd, 11.6, 4.8)
3.54 (1H, dd, 11.6, 3.2)
3.94 (1H, m)
3.35 (1H, dd, 4.8, 4.1)
3.31 (1H, dt, 5.1, 4.8)
1.52 (2H, m)
2
3
4
51.7
75.2
72.0
32.9
1,3,4,10
1,2,4,5
2,3,5,6
3,4,6
5,17,18,
200,140
H-2/H-1,3
H-3/H-2,4
H-4/H-3,5
H-5/H-4,6
H-6,17/H-5,18;
H-50,100/H-40,
H-110; H-150,190/
H-140,200
H-18,200/H-17,190
N-H/H-2
5
6–17,
50 –100,150—190
1.06 (44H, br s)
29.4-22.4
18,200
NH
10
0.68 (6H, t, 6.4)
7.42 (1H, d, 8.8)
–
3.82 (1H, d, 4.3)
3.62 (1H, dt, 6.2, 4.3)
1.58 (2H, m)
13.7
–
174.8
74.3
72.8
31.6
130.5, 129.6
32.3
17,190
1,2,3,4,10
–
10,30,40,
10,20,40,
20,30,50,
110,140,
120,130
–
20
H-20/H-30
30
H-30/H-20,40
H-40/H-30,50
H-120,130/H-110,140
H-110,140/H-120,130
40
120,130
110,140
5.21 (2H, dt, 16.5, 5.0)
1.81 (4H, m)
325
237
139
13'
85
O
OH
OH
340
20'
CH3
3'
1'
2'
12'
HN
267
OH
18
CH3
HO
3
2
4
1
OH
227
Figure 4. Structure and mass fragmentation pattern of 4.
were comparable with reported ceramides
having same configurations [15–17]. Based
on these evidences, nudicaulin D (4) was
assigned the structure (2S,3S,4R,)-2-
{[(2R,3S,12E)-2,3-dihydroxyeicos-12-
enoyl]amino}octadecane-1,3,4-triol.
Germany) as positive control in the
assay. The results (Table 5) showed that
compounds 1–4 were moderate inhibitors
of enzyme lipoxygenase.
Table 5. In vitro quantitative inhibition of
lipoxygenase by compounds 1–4.
Lipoxygenases (EC 1.13.11.12) are
potential target for the rational drug design
and discovery of mechanism-based inhibi-
tors for the treatment of a variety of
disorders such as bronchial asthma,
inflammation, cancer, and autoimmune
diseases. Compounds 1–4 were evaluated
for lipoxygenase inhibitory potential using
baicalein (Aldrich Chem. Co., Seelze,
Sr. no.
Compounds
IC50 ^ SEM (mM)a
1
2
3
4
5
1
2
3
4
193 ^ 1.11
105 ^ 1.34
103 ^ 1.74
163 ^ 1.25
22.4 ^ 1.3
Baicaleinb
a Standard error of the mean of five assays.
b Standard inhibitor of the lipoxygenase enzyme.

Journal of Asian Natural Products Research
551
3. Experimental
methanol. The methanolic extract was
evaporated under vacuum to a dark greenish
mass (1.2kg) which was suspended in
water and extracted with n-hexane and
ethyl acetate. The n-hexane soluble fraction
(150g) was subjected to CC over silica gel
eluting with n-hexane, n-hexane–dichlor-
omethane, dichloromethane, dichloro-
methane–methanol, and methanol in
increasing order of polarity. The sub-
fractions (fA–fI) obtained from the main
column subjected to further CC using
isocratic 15% n-hexane–dichloromethane
yielded 1-hexatriacontanol and b-sitosterol;
20% n-hexane in dichloromethane, octade-
cyl 4-hydroxycinnamate and elaidic acid;
30% n-hexane–dichloromethane, cholesta-
5,22-diene-3,7-diol and oleanolic acid; 70%
n-hexane–dichloromethane, apigenin; 1%
methanol in dichloromethane, nudicaulin
A (1); 3% methanol in dichloromethane, b-
sitosterol 3-O-b-D-glucopyranoside; 5%
methanol in dichloromethane, nudicaulin B
(2); 7% methanol in dichloromethane,
nudicaulin D (4); and 10% methanol in
3.1 General experimental procedures
Melting points were determined by a Buchi
434 melting point apparatus (Flavil, Swit-
zerland). IR spectra were recorded on
Shimadzu 460 spectrometer (Duisburg,
1
Germany). H (400, 500 MHz), 13C NMR
(100, 125 MHz), and 2D NMR (HMQC,
HMBC, and COSY; 400, 500 MHz) spectra
were recorded on Bruker spectrometer
(Zurich, Switzerland). The chemical shift
values (d) are reported in ppm and the
coupling constant (J) is in Hz. EI-MS, HR-
EI-MS, FAB-MS, and HR-FAB-MS were
recorded on Finnigan (Varian MAT, Wald-
bronn, Germany) JMS H £ 110 with a data
system and JMSA 500 mass spectrometers,
respectively. The gas chromatography (GC)
was performed on a Shimadzu gas chro-
matograph (GC-9A; Noisiel, France) (3%
OV-1 silanized chromosorb W, column
temperature 1808C, injection port and
detector temperature 275–3008C, flow rate
35ml/min, and flame-ionization detector).
Chromatographic separations were carried
out using aluminum sheets pre-coated with
silica gel 60 F254 (20 £ 20cm, 0.2 mm
thick; E. Merck; Darmstadt, Germany) for
thin layer chromatography (TLC) and silica
gel (230–400 mesh, Darmstadt, Germany)
for CC. TLC plates were visualized under
UV at 254 and 366 nm and by spraying with
ceric sulphate reagent solution (by heating).
dichloromethane, nudicaulin
respectively.
C
(3),
3.3.1 Nudicaulin A (1)
Colorless amorphous powder (49 mg). mp
24
106–1088C; ½aꢀD þ 16.9 (c 0.2, MeOH);
IR ymax (KBr) cm21: 3640, 2910, 1650,
1
1625; H and 13C NMR spectral data, see
Table 1; HR-EI-MS: m/z 665.6330 [M]þ
(calcd for C42H83NO4, 665.6322).
3.2 Plant material
The whole plant material of L. nudicaulis
was collected from Cholistan Desert
(Punjab), Pakistan in April 2008. It was
identified by Dr Muhammad Arshad, Plant
Taxonomist, Cholistan Institute for Desert
Studies (CIDS), The Islamia University of
Bahawalpur, where a voucher specimen is
deposited (0022-LN/CIDS/08).
3.3.2 Nudicaulin B (2)
White amorphous powder (35 mg). mp
24
114–1168C; ½aꢀD þ 39.7 (c 0.15 g/ml,
MeOH); IR ymax (KBr) cm21: 3334, 3215,
2919, 1622, 1602; 1H and 13C NMR spectral
data, see Table 2; HR-EI-MS: m/z 681.6280
[Mþ (calcd for C42H83NO5, 681.6271).
3.3 Extraction and isolation
3.3.3 Nudicaulin C (3)
24
The whole plant of L. nudicaulis (26 kg) was
shade dried, ground, and extracted with
Colorless gummy solid (44 mg). ½aꢀD þ
32.1 (c 0.12 g/ml, MeOH); IR ymax (KBr)

552
N. Riaz et al.
cm21: 3346, 3242, 2910, 1639, 1616;
1H and 13C NMR spectral data, see Table 3;
HR-FAB-MS (2ve mode) m/z 856.6884
[M–H]2 (calcd for C49H94NO10, 856.
6877).
3.4.2 Methyl ester derived from 2
½aꢀD 2 7.1 (c 0.01); 1H NMR (CDCl3,
24
400 MHz): d 4.12 (1H, t, J ¼ 6.9 Hz, H-20),
3.53 (3H, s, MeO), 1.98 (3H, s, MeCO),
1.17–1.27 (28H, br s, CH2-40-170), 0.83
(3H, t, J ¼ 6.7 Hz, CH3-180); GC-MS: m/z
356.
3.3.4 Nudicaulin D (4)
Colorless shiny powder (57 mg). mp 139–
24
3.4.3 Methyl ester derived from 3
1418C; ½aꢀD þ 29.9 (c 0.22, MeOH); IR
1
24
max (KBr) cm21: 3440, 3296, 2914, 1663,
½aꢀD 2 11.2 (c 0.011); H NMR (CDCl3,
400 MHz): d 4.15 (1H, t, J ¼ 6.6 Hz, H-20),
3.56 (3H, s, MeO), 1.99 (3H, s, MeCO),
1.18–1.29 (26H, br s, CH2-40-160), 0.86
(3H, t, J ¼ 6.9 Hz, CH3-170); GC-MS: m/z
342.
y
1616; H and 13C NMR spectral data, see
Table 4; HR-EI-MS: m/z 641.5600 [M]þ
(calcd for C38H75NO6, 641.5594).
1
3.4 Methanolysis
Compounds 1–4 (12 mg each) were
refluxed separately with 6 ml of 1N HCl
and 25 ml of MeOH for 15 h. The reaction
mixture was then extracted with n-hexane
to obtain the corresponding fatty acid
methyl esters, which were analyzed by GC-
MS after acetylation with aceticanhy-
dride–pyridine. The aqueous layer of 1,
2, and 4 was evaporated, and the residue
was acetylated. Purification over Sephadex
LH-20 and elution with CH2Cl2/MeOH
(1:1) gave the corresponding acetylated
sphingosines, which were analyzed by GC-
MS. The aqueous layer of 3 was evaporated
to dryness, and the residue was separated
by silica gel CC as sphingosine base and
methylated sugar. The base was acetylated
and analyzed by GC-MS. The sugar was
identified as methyl b-D-glucopyranoside
based on the sign of optical rotation
[a]D þ 76.2 (c 0.1, MeOH) and Co-TLC
profile (Rf 0.45 (EtOAc/MeOH/H2O;
5:2:0.5)).
3.4.4 Methyl ester derived from 4
1
24
½aꢀD þ 9.6 (c 0.012); H NMR (CDCl3,
400 MHz): d 5.28 (2H, dt, J ¼ 16.0, 5.6 Hz,
H-12,13), 4.19 (1H, d, J ¼ 4.3 Hz, H-20),
4.11 (1H, dt, J ¼ 6.1, 4.3 Hz, H-30), 3.50
(3H, s, MeO), 2.00 (6H, s, 2 £ MeCO),
1.15–1.25 (22H, br s, CH2-50-100,150-190),
0.90 (3H, t, J ¼ 6.5 Hz, CH3-200); GC-MS:
m/z 440.
3.4.5 Acetylsphingamine derived from 1
1
24
½aꢀD þ 11.2 (c 0.01); H NMR (CDCl3,
400 MHz): d 8.06 (1H, d, J ¼ 7.6 Hz, NH),
5.31 (2H, dt, J ¼ 15.1, 5.2 Hz, H-14,15),
4.56 (1H, dd, J ¼ 5.1, 4.3 Hz, H-4), 4.50
(1H, m, H-2), 4.42 (1H, dd, J ¼ 10.3,
5.6 Hz, H-1), 4.33 (1H, dd, J ¼ 10.3,
3.3 Hz, H-1), 4.17 (1H, dd, J ¼ 5.1,
3.2 Hz, H-3), 2.00 (6H, 2 £ MeCO), 1.98
(6H, 2 £ MeCO), 1.13–1.21 (26H, br s,
CH2-7-12,17-23), 0.89 (t, J ¼ 6.5 Hz, CH3-
24); GC-MS: m/z 567.
3.4.1 Methyl ester derived from 1
3.4.6 Acetylsphingamine derived from 2
1
1H NMR (CDCl3, 400 MHz): d 3.51 (s,
MeO), 2.32 (2H, t, J ¼ 6.5 Hz, H-20), 1.51
(2H, m, H-30), 1.19–1.28 (28H, br s, CH2-
40-170), 0.84 (3H, t, J ¼ 6.8 Hz, CH3-180);
GC-MS: m/z 298.
½aꢀD þ 19.1 (c 0.015); H NMR (CDCl3,
400 MHz): d 7.96 (1H, d, J ¼ 7.9 Hz, NH),
5.33 (2H, dt, J ¼ 15.5, 5.5 Hz, H-14,15),
4.66 (1H, dd, J ¼ 5.0, 4.1 Hz, H-4), 4.55
(1H, m, H-2), 4.44 (1H, dd, J ¼ 10.2,
24

Journal of Asian Natural Products Research
553
5.4 Hz, H-1), 4.33 (1H, dd, J ¼ 10.2, 3.1 Hz,
H-1), 4.19 (1H, dd, J ¼ 5.0, 3.1 Hz, H-3),
2.00 (12H, 4 £ MeCO), 1.15–1.25 (26H, br
s, CH2-7-12,17-23), 0.85 (t, J ¼ 6.6 Hz,
CH3-24); GC-MS: m/z 567.
solution of diazomethane and analyzed
by GC-MS.
3.6 Lipoxygenase assay
The lipoxygenase activity was assayed
according to the method of Tappel et al.
[24] with slight modifications. A total
volume of 200ml assay mixture contained
160 ml sodium phosphate buffer (100 mM,
pH 8.0), 10ml of test compound, and 20ml
purified lipoxygenase (Sigma Chemicals,
Seelze, Germany). The contents were pre-
incubated for 10 min at 258C. The reaction
was initiated by addition of 10 ml linoleic
acid (substrate solution). The change in
absorbance was observed after 6 min at
234 nm. All reactions were performed in
triplicates in 96-well microplate reader
(Synergy HT, Biotek, Winooski, Vermont,
USA). The positive and negative controls
were included in the assay. The percentage
inhibition (%) was calculated by formula:
3.4.7 Acetylsphingamine derived from 3
1
24
½aꢀD þ 19.1 (c 0.013); H NMR (CDCl3,
400 MHz): d 7.90 (1H, d, J ¼ 8.2 Hz, NH),
5.29 (2H, dt, J ¼ 16.1, 5.8 Hz, H-14,15),
4.60 (1H, dd, J ¼ 5.1, 4.0 Hz, H-4), 4.52
(1H, m, H-2), 4.40 (1H, dd, J ¼ 10.6,
5.3 Hz, H-1), 4.30 (1H, dd, J ¼ 10.6, 3.0 Hz,
H-1), 4.16 (1H, dd, J ¼ 5.1, 3.2 Hz, H-3),
2.01 (12H, 4 £ MeCO), 1.16–1.26 (30H, br
s, CH2-7-12,17-25), 0.87 (t, J ¼ 6.2 Hz,
CH3-26); GC-MS: m/z 595.
3.4.8 Acetylsphingamine derived from 4
1
24
½aꢀD þ 16.9 (c 0.012); H NMR (CDCl3,
400 MHz): d 8.16 (1H, d, J ¼ 7.9 Hz, NH),
4.56 (1H, dd, J ¼ 5.2, 3.9 Hz, H-4), 4.50
(1H, m, H-2), 4.39 (1H, dd, J ¼ 11.0,
5.1 Hz, H-1), 4.30 (1H, dd, J ¼ 11.0, 3.0 Hz,
H-1), 4.15 (1H, dd, J ¼ 5.2, 3.0 Hz, H-3),
2.00 (12H, 4 £ MeCO), 1.19–1.30 (24H, br
s, CH2-6-17), 0.86 (t, J ¼ 6.5 Hz, CH3-18);
GC-MS: m/z 485.
Inhibition ð%Þ ¼ ðcontrol 2 testÞ £ 100:
Control is the total enzyme activity without
inhibitor and test is the activity of the test
compound. IC50 values (concentration at
which there is 50% enzyme inhibition) of
selected samples were calculated using EZ-
Fit Enzyme kinetics software (Perrella
Scientific Inc. Amherest, MA, USA).
3.5 Oxidative cleavage of the double
bond in 1–4
Separately, to the solution of acetyl-
sphingamines of compounds 1–3 and the
methyl ester derived from 4 (4 mg each) in
acetone, 1 ml of 0.04 M solution of K2CO3,
6 ml of an aqueous solution of 0.025 M
KMnO4, and 0.09 M NaIO4 were added in
100 ml round-bottomed flask. The reaction
was allowed to proceed at 378C for 18 h.
After acidification with 5 N H2SO4, the
solution was decolorized with a 1 M
solution of oxalic acid and extracted with
Et2O (3–10 ml). The combined organic
extracts were dried over Na2SO4, filtered,
and concentrated. The resulting carboxylic
acids were methylated with ethereal
Acknowledgments
The authors are thankful to Higher Education
Commission (HEC) Pakistan and Alaxander
von Humboldt (AvH) Foundation, Germany for
financial support. We are also obliged to Third
World Academy of Science (TWAS), Italy, for
providing some of the basic laboratory facilities
in Chemistry Department of IUB.
References
[1] P.P.J. Herman, E. Retief, M. Koekemoer,
and W.G. Welman, Strelitzia 10, 101
(2000).
[2] K.R. Prabhu and V. Venkateswarlu,
J. Indian Chem. Soc. 46, 176 (1969).

554
N. Riaz et al.
[3] S. Rashid, M. Ashraf, S. Bibi, and
[15] H.C. Kwon, K.C. Lee, O.R. Cho, Y. Jung,
S.Y. Cho, S.Y. Kim, and K.R. Lee, J. Nat.
Prod. 66, 466 (2003).
[16] V.U. Ahmad, M. Zubair, M.A. Abbasi,
F. Kousar, M.A. Rashid, N. Rasool, and
R.B. Tareen, Nat. Prod. Res. 20, 69
(2006).
[17] P. Muralidhar, M.M. Kumar, N. Krishna,
C.B. Rao, and D.V. Rao, Chem. Pharm.
Bull. 53, 168 (2005).
[18] E. Ahmed, A. Malik, N. Afza, N. Riaz,
I. Anis, A. Sharif, S. Farheen, M.A. Lodhi,
and M.I. Choudhary, Nat. Prod. Res. 21,
69 (2007).
[19] H.S. Garg and S. Agrawal, J. Nat. Prod.
58, 442 (1995).
[20] P. Muralidhar, N. Krishna, M.M. Kumar,
C.B. Rao, and D.V. Rao, Chem. Pharm.
Bull. 51, 1193 (2003).
[21] T. Natori, M. Morita, K. Akimoto, and
Y. Koezuka, Tetrahedron 50, 2771
(1994).
[22] N. Riaz, S.A. Nawaz, N. Mukhtar,
A. Malik, N. Afza, S. Ali, S. Ullah,
P. Muhammad, and M.I. Choudhary,
Chem. Biodiver. 4, 72 (2004).
R. Anjum, Pak. J. Biol. Sci. 3, 808 (2000).
[4] A. Krishnamurthi, The Wealth of India
(Council of Scientific and Industrial
Research (CSIR), New Delhi, 1969),
Vol. 1, p. 68.
[5] E. Nasir and S.I. Ali, Flora of West
Pakistan (Fakhri Printing Press, Karachi,
1972), Vol. 712.
[6] S.R. Baquar, Medicinal and Poisonous
Plants of Pakistan (Printas Printing Press,
Karachi, 1989), Vol. 31&258.
[7] M.M. Gupta, R.K. Verma, and S.C. Singh,
Fitoterapia 60, 476 (1989).
[8] R.N. Yadava and N. Chakravarti,
Indian J. Chem. Section B 48B, 83 (2009).
[9] R.M.A. Mansour, A.A. Ahmed, and
N.A.M. Saleh, Phytochemistry 22, 2630
(1983).
[10] F. Moussaoui, A. Zellagui, N. Segueni,
A. Touil, and S. Rhouati, Rec. Nat. Prod.
4, 91 (2010).
[11] D. Ali, S.M.S. Hussain, A. Malik, and
Z. Ahmed, J. Chem. Soc. Pak. 25, 341
(2003).
[12] A.A. El-Bassuony and A.M. Kabbash,
Pharm. Mag. 4, 249 (2008).
[23] W. Jin, K.L. Rinehart, and E.A. Jares-
Erijman, J. Org. Chem. 59, 144 (1994).
[24] A.L. Tappel, Arch. Biochem. Biophy. 44,
378 (1953).
[13] M.M. Bhandari, Flora of Indian Desert
(Mps Repros, Jodhpur, 1988), p. 182.
[14] S. Rashid, M. Ashraf, S. Bibi, and
R. Anjum, Pak. J. Biol. Sci. 3, 630 (2000).