New lipoxygenase and cholinesterase inhibitory sphingolipids from Carthamus oxyacantha

Article abstract of DOI:10.1080/14786419.2015.1075524

Two new sphingolipids: oxyacanthin A [(2S,3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol; 1] and B [(2S,3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol-1-O-β-D-glucopyranoside; 2], together with 1-octacosanol, β-sitosterol, β-sitosterol 3-O-β-D-glucopyranoside and luteolin 7-O-β-glucopyranoside were isolated from the methanolic extract of the whole plant of Carthamus oxyacantha. Their structures were elucidated using¹H and¹³C 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 in comparison with the literature data of the related compounds. Both the compounds 1 and 2 showed inhibitory potential against lipoxygenase (LOX) in a concentration-dependent manner with IC₅₀values 83.3?±?1.3 and 245.7?±?1.1?μM, whereas compound 2 showed inhibition against enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC₅₀values 65.3?±?0.1 and 93.6?±?0.1?μM, respectively.

Full text of DOI:10.1080/14786419.2015.1075524

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New lipoxygenase and cholinesterase
inhibitory sphingolipids from
Carthamus oxyacantha
Muhammad Dilshada, Naheed Riaza, Muhammad Saleema, Nusrat
Shafiqa, Muhammad Ashrafab, Tayaba Ismailb, Hafiza Mehwish
Rafiqa & Abdul Jabbara
a Department of Chemistry, The Islamia University of Bahawalpur,
Bahawalpur, Pakistan
b Department of Biochemistry and Biotechnology, The Islamia
University of Bahawalpur, Bahawalpur, Pakistan
Published online: 19 Aug 2015.
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To cite this article: Muhammad Dilshad, Naheed Riaz, Muhammad Saleem, Nusrat Shafiq,
Muhammad Ashraf, Tayaba Ismail, Hafiza Mehwish Rafiq & Abdul Jabbar (2015): New lipoxygenase
and cholinesterase inhibitory sphingolipids from Carthamus oxyacantha, Natural Product Research:
Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1075524
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Natural Product research, 2015
http://dx.doi.org/10.1080/14786419.2015.1075524
New lipoxygenase and cholinesterase inhibitory
sphingolipids from Carthamus oxyacantha
Muhammad Dilshada, Naheed Riaza, Muhammad Saleema, Nusrat Shafiqa,
Muhammad Ashrafab, Tayaba Ismailb, Hafiza Mehwish Rafiqa and Abdul Jabbara
adepartment of chemistry, the Islamia university of Bahawalpur, Bahawalpur, Pakistan; bdepartment of
Biochemistry and Biotechnology, the Islamia university of Bahawalpur, Bahawalpur, Pakistan
ARTICLE HISTORY
received 9 april 2015
accepted 16 July 2015
ABSTRACT
Two new sphingolipids: oxyacanthin A [(2S,3S,4R)-2-{[(2R,5E)-2-
hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol;1] and B [(2S,-
3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]amino}hexaei-cosane
1,3,4-triol-1-O-β-D-glucopyranoside; 2], together with 1-octacosanol,
β-sitosterol, β-sitosterol 3-O-β-D-glucopyranoside and luteolin 7-O-
β-glucopyranoside were isolated from the methanolic extract of the
whole plant of Carthamus oxyacantha. Their structures were elucidated
KEYWORDS
carthamus oxyacantha;
sphingolipids; secondary
metabolites; lipoxygenase
inhibition; cholinesterase
inhibition
1
using H 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 in comparison with
the literature data of the related compounds. Both the compounds
1 and 2 showed inhibitory potential against lipoxygenase (LOX) in
a concentration-dependent manner with IC50 values 83.3 1.3 and
245.7 1.1 µM, whereas compound 2 showed inhibition against
enzymes acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) with IC50 values 65.3 0.1 and 93.6 0.1 µM, respectively.
O
OH3'
17'
7'
5'
18'
6'
1'
HN
CH3
4'
2'
OH
26
RO
CH3
2
3
4
25
1
OH
1 R = H
2 R = Glucose
1. Introduction
The genus Carthamus of the plant family ‘Asteraceae’ comprises 25 species which are very
important as pharmaceutical point of view and have commercial values as well (Hacioglu
et al. 2012;Talebi et al. 2012). Carthamus tinctorius is used for the treatment of cough, typhoid
CONTACT Naheed riaz
nrch322@yahoo.com, nrch322@iub.edu.pk
supplementary material relating to this article is available online at http://dx.doi.org/10.1080/14786419.2015.1075524
(s1–s25).
© 2015 taylor & Francis

2
M. DILSHAD ET AL.
fever, throat disorders and as herbal tea to reduce blood cholesterol levels (Khan & Khatoon
2008). It is also used to get relief from cardiovascular problems, swelling, menstrual disorders
and pain (Arpornsuwan et al. 2010). It shows anti-apoptotic, anti-inflammatory, antioxidant
and anti-thrombotic activities (Jun et al. 2011). In China, Japan, India and Korea, it is used
as herbal medicine for the treatment of inflammation, gynecological disorders and heart
problems (Arpornsuwan et al. 2012). It also acts as neuroprotective, calcium antagonist and
anti-coagulant (Han et al. 2009). The decoction of C. tinctorius is used to reduce blood vis-
cosity (Li et al. 2009). The oil of its seeds contains linoleic acid and various amount α-tocoph-
erol, β-tocopherol and γ-tocopherol which are helpful in reducing blood cholesterol levels
(Matthaus et al. 2015). The extract of C. lanatus possesses anti-inflammatory, anti-microbial
and anti-analgesic activities (Topashka-Ancheva et al. 2006) and also used traditionally as sed-
ative and anti-tumour agent (Taskova et al. 2002; Bocheva et al. 2003). Carthamus oxyacantha
is found in dry places and plains of Pakistan, Afghanistan, Turkmenistan, India, Azerbaijan,
Iraq, Tajikistan, Kyrgyzstan and Iran. The flowers of C. oxyacantha are used as herbal remedy
in rheumatism, cerebral thrombosis, bronchitis, male infertility and jaundice, while its seeds
have laxative property and oil is used in scabies therapy. It is used to reduce pain and swell-
ing in injuries, exhibits labour induction property, increases blood circulation (Ahmad et al.
2007–2010) and has anti-hyperlipidemic character (Ahmad et al. 2009). The diverse biological
activities of C. oxyacantha motivated us to explore this plant for its bioactive constituents.
Herein, we report the isolation and characterisation of two new sphingolipids (1,2) (Figure 1)
together with 1-octacosanol (Safder et al. 2009), β-sitosterol (Rubinstein et al. 1976; Bernard &
Eokes 1977; Holland et al. 1978), β-sitosterol 3-O-β-D-glucopyranoside (Rubinstein et al. 1976;
Bernard & Eokes 1977; Holland et al. 1978) and luteolin 7-O-β-glucopyranoside (Wang et al.
1998) from the methanolic extract of the whole plant of C. oxyacantha.
Sphingolipids are the waxy lipids composed of sphingosine and a fatty acid generally found
in high concentrations within the cell membrane of cells. They are one of the component
lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer. Sphingolipids
can participate in a variety of cellular signalling including regulating differentiation, prolif-
eration and programmed cell death of cells. They are implicated in a variety of physiological
functions including apoptosis, cell growth arrest, differentiation, cell senescence, cell migra-
tion and adhesion. Roles for these chemicals and their downstream metabolites have also
been suggested in a number of pathological states including cancer, neurodegeneration,
diabetes, microbial pathogenesis, obesity and inflammation (Hannun & Obeid 2008).
O
OH3'
17'
7'
5'
18'
6'
1'
HN
CH3
4'
2'
OH
26
RO
CH3
2
3
4
25
1
OH
1 R = H
2 R = Glucose
Figure 1. structures of compounds 1 and 2 isolated from C. oxyacantha.

NATuRAL PRODuCT RESEARCH
3
Lipoxygenases (LOX, EC 1.13.11.12) are potential targets for the rational drug design and
discovery of mechanism-based inhibitors for the treatment of a variety of disorders such as
bronchial asthma, inflammation, cancer and autoimmune diseases. Cholinesterase enzymes
(AChE, BChE) are implicated as key biological players in Alzheimer’s disease, which makes
them logical targets for inhibitory therapeutics. Compounds 1 and 2 were evaluated for LOX,
AChE and BChE inhibitory potential using baicalein and eserine (Aldrich Chem. Co.; Seelze,
Germany) as positive controls in the assays. The results (Table 1) showed that compounds
1 and 2 were moderate inhibitors of LOX, whereas compound 2 showed moderate to weak
inhibitory potential against AChE and BChE.
2. Results and discussion
Compound 1 was isolated as colourless amorphous powder. Its IR spectrum showed the
absorption bands at 3500 (O–H), 3450 (N–H), 1670 (C=O) and 1650 (C=C) cm−1. The HR-EI-MS
showed the molecular ion peak [M]+ at m/z 709.6567 corresponding to the molecular formula
C44H87NO5. The 1H NMR spectrum of 1 afforded a signal at δH 7.80 (d, J = 8.8 Hz, 1H) was attrib-
uted to an amide function. Two olefinic protons were observed as a broad multiplet at δH 5.32
(br m, 2H). The multiplicity of this signal at half height was calculated to be 22.5 Hz. The W1/2
(22.5 Hz) and the chemical shifts of C-4′ and C-7′ (δ 35.5, 33.8) adjacent to the double bond
indicated its trans geometry of the double bond (Shibuya et al. 1990). A methine resonating at
δH 3.91 (m, 1H) was correlated in HSQC spectrum with a carbon at δC 51.5 revealed its attach-
ment with the amide function. Furthermore, the same spectrum showed three oxymethines at
δH 3.82 (dd, J = 6.6, 3.4 Hz, 1H), 3.46 (dd, J = 4.1, 3.7 Hz, 1H), 3.38 (m, 1H) and an oxymethylene
at δH 3.63 (dd, J = 11.5, 3.8 Hz, 1H) and 3.57 (dd, J = 11.5, 5.0 Hz, 1H). The 13C NMR spectrum of
1 (Table 1) displayed signals at δC 173.2 and 131.4 for carbonyl and two olefinic carbons, three
oxymethines at δC 75.1 (C-3), 72.9 (C-2′) and 72.7 (C-4) and an oxymethylene at δC 60.6 (C-1),
respectively. Another methine carbon resonating at δC 51.5 (C-2) was attested for an azome-
thine function. These values were similar to the data reported for ceramides (Kwon et al. 2003;
Gao et al. 2004; Riaz et al. 2012) concluding 1 to be a ceramide. The lengths of fatty acid and
amine chains could be determined due to mass spectrometry and/or chemical degradation.
The length of the fatty acid chain was found to be composed of eighteen carbons due to a
fragment ion at m/z 281 in EI-MS spectrum containing double bond and a hydroxyl group.
The position of the double bond could be determined due to COSY spectrum, fragment
ions at m/z 195 and 169 and HMBC correlations. On the other hand, the length of the amine
chain was also determined due to fragment ion in EI-MS spectrum at m/z 428. Two hydroxyl
groups were confirmed at C-3 and C-4 due to COSY correlations of azomethine (δH 3.91, H-2)
with oxymethylene (δH 3.63, 3.57, H-1) and oxymethine (δH 3.46, H-3) which further correlated
with H-4 (δH 3.38). Methanolysis of 1 followed by acetylation and GC–MS analysis indentified
Table 1. ache, Bche and loX inhibition studies of compounds 1 and 2.
Conc.
mM
AChE
% inhibition
BChE
% inhibition
LOX
% inhibition
Sr. no.
IC50 (µM)
IC50 (µM)
IC50 (µM)
1
0.5
0.5
0.5
0.5
7.6 0.1
84.1 0.3
91.3 1.1
–
–
16.7 0.1
58.1 0.2
82.8 1.1
–
–
93.6 0.1
0.6 0.0001
–
84.2 1.6
57.8 1.2
–
83.3 1.3
245.7 1.1
–
2
65.3 0.1
0.04 0.001
–
eserine
Baicalein
93.8 1.3
22.4 1.3
Notes: compounds were dissolved in methanol and assayed at concentration of 0.5 mM.
results are presented as mean sem, n = 3.

4
M. DILSHAD ET AL.
the fatty acid and amine chains as methyl 2-acetoxyoctadec-5-enoate (m/z 354) and 2-aceta-
mino-1,3,4-triacetoxyhexaeicosane (m/z 597), respectively. Position of the double bond was
further confirmed through its cleavage by periodate (Ahmed et al. 2007). It is reported in the
literature that both the optical rotation and the chemical shifts of both the 1H and 13C atoms
at the stereogenic centres provide a strong evidence for the absolute configuration of sphin-
golipids. Since most of the spectra of sphingolipids have so far been reported in pyridine,
therefore, the 1H NMR spectrum of 1 was repeated in pyridine, and the chemical shifts of the
H-atoms attached to the stereogenic were found to be δH 4.57 (dd, J = 10.4, 5.3 Hz, H-1a, 1H),
4.40 (dd, J = 10.4, 3.1 Hz, H-1b, 1H), 5.08 (m, H-2, 1H), 4.32 (dd, J = 4.7, 3.2 Hz, H-3, 1H), 4.25
(td, J = 5.5, 4.7, Hz, H-4, 1H) and 4.56 (t, J = 6.5 Hz, H-2′, 1H), whereas the carbons shifts of
the respective centres were appeared at δC 61.4, 52.0, 75.5, 72.4 and 72.7, respectively, which
were in complete agreement with those of sphingolipids with a 2S,3S,4R,2R′ configuration
(Muralidhar et al. 2005; Kang et al. 2007; Riaz et al. 2007; Dongfack et al. 2012). The optical
rotations of 1 ([α]D=+41.0) and its methanolysis products ([α] = 8.1 and +18.9) which were
comparable with those of sphingolipids with a 2S,3S,4R,2′DR configurations (Muralidhar
et al. 2005; Kang et al. 2007; Dongfack et al. 2012). Based on the above data and discussion,
compound 1 was finally characterised as (2S,3S,4R)-2-{[(2R,5E)-2-hydroxyoctadec-5-enoyl]
amino}hexaeicosane-1,3,4-triol.
Compound 2 was isolated as colourless gummy solid. Its IR spectrum displayed absorp-
tion band at 3505 (O–H), 3440, 1670 (amide) and 1655 cm−1 (C=C) similar to that of 1. The
HR-FAB-MS showed molecular ion peak [M–H]+ at m/z 870.7021 gave molecular formula
C50H96NO10. The 1H and 13C NMR spectra of 2 were very similar to that of 1 containing the usual
signals for ceramide moiety, with the addition of an anomeric methine at δH 4.28 (d, J = 7.5 Hz,
H-1″, 1H) indicating the presence of a sugar moiety in 2. The remaining signals for sugar unit
were observed at δH 3.85 (dd, J = 10.4, 4.5 Hz, H-6″a, 1H), 3.71 (dd, J = 10.4, 2.9 Hz, H-6″b, 1H),
3.79 (m, H-5″, 1H), 3.65 (m, H-4″, 1H), 3.39 (m, H-2″, 1H) and 3.36 (m, H-3″, 1H). The 13C NMR
spectrum of 2 also provided evidence for ceramide glycoside as it displayed anomeric carbon
at δC 104.7 (C-1″) with other sugar signals at δC 78.0 (C-5″), 77.9 (C-2″), 72.9 (C-3″), 71.5 (C-4″)
and a methylene carbon at δC 62.6 (C-6″) (Riaz et al. 2007, 2012). The attachment of sugar
could be confirmed at C-1 due to its downfield shift at δC 69.9 and HMBC correlation of H-1″
(δH 4.28) with C-1 (δH 69.9). The length of fatty acid and amine chains, position of the double
bond and hydroxyl groups were found similar as in 1 by comparing the mass fragmentation
patterns, COSY spectrum and HMBC correlation and by chemical methods (methanolysis
and periodate cleavage) of 2 and 1, respectively. The stereochemistry at various stereogenic
centres was found similar to those for 1. Based on the above data and discussion, com-
pound 2 has been found to be glucoside of 1 and characterised as [(2S,3S,4R)-2-{[(2R,5E)-2-
hydroxyoctadec-5-enoyl]amino}hexaeicosane-1,3,4-triol-1-O-β-d-glucopyranoside.
3. Experimental
3.1. General experimental procedures
Infrared (IR) spectra were recorded on Shimadzu 460 spectrometer (Duisburg, Germany). 1H
NMR, HMQC, HMBC and COSY spectra were acquired on either a 400 or 500 MHz, whereas
the 13C NMR spectra were recorded on a 100 or 125 MHz on Bruker spectrometer (Zurich,
Switzerland). The chemical shift values (δ) 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)
JMS H × 110 with a data system and JMSA 500 mass spectrometers (Varian MAT, Waldbronn,

NATuRAL PRODuCT RESEARCH
5
Germany), respectively. The gas chromatography (GC) was performed on a Shimadzu gas
chromatograph (GC-9A; Noisiel, France) (3% OV-1 silanised chromosorb W, column tem-
perature 180 °C, injection port and detector temperature 275–300 °C, flow rate 35 mL/min,
flame-ionisation detector). Chromatographic separations were carried out using aluminium
sheets pre-coated with silica gel 60 F254 (20 cm × 20 cm, 0.2 mm thick; E. Merck; Darmstadt,
Germany) for thin layer chromatography (TLC) and silica gel (230–400 mesh; Darmstadt,
Germany) for column chromatography. TLC plates were visualised under uV at 254 and
366 nm and by spraying with ceric sulphate reagent solution (by heating).
3.2. Plant material
The whole plant of C. oxyacantha M. Bieb. (05 kg dry weight) was collected from the fields
opposite to Baghdad-ul-Jadeed Campus, The Islamia university of Bahawalpur, Bahawalpur,
Pakistan, in May 2012 and was identified by Mr. Muhammad Waris, Plant Taxonomist,
Cholistan Institute for Desert Studies, The Islamia university of Bahawalpur, Bahawalpur,
Pakistan, where a voucher specimen is deposited (CO-129/12).
3.3. Extraction and isolation
The shade dried whole plant material of C. oxyacantha was extracted thrice in methanol
(3 L × 20 L) at room temperature and concentrated on rotary evaporator at 35 °C to a dark
gummy mass (150 g) which was subjected to silica gel column chromatography and eluted
with n-hexane, n-hexane-dichloromethane (DCM), DCM, DCM-methanol and methanol in
increasing order of polarity to get seven fractions (F1–F7). The fraction F1 obtained using
n-hexane:DCM (8:2) was further purified using n-hexane:DCM (7.5:2.5) yielded 1-octacos-
anol (20 mg). The fraction F2 obtained with solvent system n-hexane:DCM (7:3) which on
further purification using n-hexane:DCM (6.5:3.5) resulted in the purification of β-sitos-
terol (54 mg). The fraction F3 obtained using 80% DCM in n-hexane, subjected to repeated
CC, on elution with 85% DCM in n-hexane gave oxyacanthin A (1) (35 mg). The fraction F4
obtained DCM:methanol (8.8:0.2) on further purification with 3% methanol in DCM afforded
β-sitosterol 3-O-β-d-glucopyranoside (60 mg). The fraction F5 got using 6% DCM:methanol
(9.4:0.6) subject on further purification by CC using methanol in DCM (9.3:0.7) afforded lute-
olin-7-O-β-glucopyranoside (42 mg). The fraction F6 was obtained using 10% methanol in
DCM, on further elution with 11% methanol in DCM oxyacanthin B (2) (44 mg).
3.3.1. Oxyacanthin A (1)
Colourless amorphous powder (35 mg);[ꢀ]24 + 41.0 (c 0.0014 g/mL, MeOH); IR (KBr cm−1) νmax
:
D
3500, 3450, 2928, 1670, 1650; HR-EI-MS: m/z 709.6567 [M]+ (calcd for C44H87NO5, 709.6584);
1H NMR data (CDCl3 + CD3OD; 500 MHz), δ: 3.63 (1H, dd, J = 11.5, 3.8 Hz, H-1a), 3.57 (1H, dd,
J = 11.5, 5.0 Hz, H-1b), 3.91 (1H, m, H-2), 3.46 (1H, dd, J = 4.1, 3.7 Hz, H-3), 3.38 (1H, m, H-4),
1.61 (4H, m, H-5,3′), 1.08 (60H, br s, H-6-25,8′-17′), 0.71 (6H, t, J = 6.5, Hz, H-26,18′), 7.80 (1H,
d, J = 8.8 Hz, N–H), 3.82 (1H, dd, J = 6.6, 3.4, Hz, H-2′), 1.54 (4H, m, H-4′,7′), 5.32 (2H, br m,
H-5′,6′). 13C NMR data (CDCl3 + CD3OD; 125 MHz), δ: 60.6 (C-1), 51.5 (C-2), 75.1 (C-3), 72.7 (C-4),
33.7 (C-5), 29.1–30.2 (C-6-25, 8′–17′), 14.5 (26, 18′), 173.2 (C-1′), 72.9 (C-2′), 35.7 (C-3′), 35.5
(C-4′), 131.4 (C-5′,6′), 33.8 (C-7′).
3.3.2. Oxyacanthin B (2)
Colourless gummy solid (44 mg);[ꢀ]24 + 33.5 (c 0.0012 g/mL, MeOH); IR (KBr cm−1) νmax: 3505,
D
3440, 2919, 1670, 1655; HR-FAB-MS (-ve mode) m/z 870.7021 [M–H]+ (calcd for C50H96NO10,

6
M. DILSHAD ET AL.
870.7034). 1H NMR data (CD3OD; 500 MHz), δ: 4.04 (1H, dd, J = 11.6, 3.9 Hz, H-1a), 3.87 (1H,
dd, J = 11.6, 5.1 Hz, H-1b), 4.24 (1H, m, H-2), 3.81 (1H, dd, J = 4.2, 3.9 Hz, H-3), 3.60 (1H, m,
H-4), 1.61 (4H, m, H-5,3′), 1.08 (60H, br s, H-6-25,8′–17′), 0.71 (6H, t, J = 6.4 Hz, H-26,18′), 7.82
(1H, d, J = 8.7 Hz, N–H), 4.06 (1H, dd, J = 6.4, 3.6 Hz, H-2′), 1.54 (4H, m, H-4′,7′), 5.41 (2H, br m,
H-5′,6′), 4.28 (1H, d, J = 7.5 Hz, H-1″), 3.39 (1H, m, H-2″), 3.36 (1H, m, H-3″), 3.65 (1H, m, H-4″),
3.79 (1H, m, H-5″), 3.85 (1H, dd, J = 10.4, 4.5 Hz, H-6″a), 3.71 (1H, dd, J = 10.4, 2.9 Hz, H-6″b).
13C NMR data (CD3OD; 125 MHz), δ: 69.9 (C-1), 51.6 (C-2), 75.5 (C-3), 73.0 (C-4), 33.7 (C-5),
29.1–30.2 (C-6-25,8′-17′), 14.5 (C-26,18′), 177.1 (C-1′), 72.9 (C-2′), 35.7 (C-3′), 35.5 (C-4′), 131.4
(C-5′,6′), 33.8 (C-7′), 104.7 (C-1″), 77.9 (C-2″), 72.9 (C-3″), 71.5 (C-4″), 78.0 (C-5″), 62.6 (C-6″).
3.4. Methanolysis
Compounds 1 and 2 (10 mg each) were refluxed separately with 5 mL of 1 N HCl and 20 mL
of MeOH for 15 h. The reaction mixture was then extracted with n-hexane to obtain the cor-
responding fatty acid methyl esters, which were analysed by GC–MS after acetylation with
acetic anhydride-pyridine. The aqueous layer of 2 was evaporated to dryness, and the residue
was separated by silica gel column chromatography as sphingosine base and methylated
sugar. The base was acetylated and analysed by GC–MS. The sugar was identified as methyl
β-d-glucopyranoside based on the sign of optical rotation [α]D + 77.1 (c 0.001, MeOH) and
Co-TLC profile (Rf 0.45 (EtOAc/MeOH/H2O; 5:2:0.5)).
3.4.1. Methyl ester derived from 1
[ꢀ]24 8.1 (c 0.001); 1H NMR (CDCl3; 400 MHz), δ: 5.31 (2H, dt, J = 15.2, 5.4, H-5′,6′), 4.11 (1H, t,
D
J = 6.4, H-2′), 3.51 (3H, s, MeO), 2.13 (4H, m, H-4′,7′), 1.98 (3H, s, MeCO), 1.19–1.29 (20H, br s,
CH2-8′–17′), 0.85 (3H, t, J = 6.5, CH3-18′); GC–MS: m/z 354.
3.4.2. Methyl ester derived from 2
[ꢀ]24 11.2 (c 0.0011); 1H NMR (CDCl3; 400 MHz), δ: 5.30 (2H, dt, J = 16.0, 5.5, H-5′,6′), 4.17 (1H, t,
D
J = 6.5, H-2′), 3.54 (3H, s, MeO), 2.12 (4H, m, H-4′,7′), 2.00 (3H, s, MeCO), 1.20–1.31 (20H, br s,
CH2-8′–17′), 0.87 (3H, t, J = 6.7, CH3-18′); GC–MS: m/z 354.
3.4.3. Acetylsphingamine derived from 1
[ꢀ]24 + 18.9 (c 0.0014); H NMR (CDCl3; 400 MHz), δ: 7.92 (1H, d, J = 7.7, NH), 4.64 (1H, dd,
1
D
J = 5.1, 4.0, H-4), 4.54 (1H, m, H-2), 4.43 (1H, dd, J = 10.1, 5.4, H-1), 4.33 (1H, dd, J = 10.1, 3.1,
H-1), 4.19 (1H, dd, J = 5.1, 3.1, H-3), 2.00 (12H, 4 × MeCO), 1.16–1.27 (40H, br s, CH2-6-25), 0.86
(t, J = 6.5, CH3-26); GC–MS: m/z 597.
3.4.4. Acetylsphingamine derived from 2
[ꢀ]24 + 19.6 (c 0.012); 1H NMR (CDCl3; 400 MHz), δ: 7.91 (1H, d, J = 8.0, NH), 4.61 (1H, dd, J = 5.0,
D
4.1, H-4), 4.52 (1H, m, H-2), 4.40 (1H, dd, J = 10.6, 5.2, H-1), 4.30 (1H, dd, J = 10.6, 3.0, H-1),
4.16 (1H, dd, J = 5.0, 3.2, H-3), 2.01 (12H, 4 × MeCO), 1.16–1.26 (40H, br s, CH2-6-25), 0.85
(t, J = 6.5, CH3-26); GC–MS: m/z 597.
3.5. Oxidative cleavage of double bond in 1 and 2
To the fatty acid methyl esters of compounds 1 and 2 (4 mg each) in acetone, added 1 mL
of 0.04 M solution of K2CO3, 6 mL of an aqueous solution 0.025 M KMnO4 and 0.09 M NaIO4
in 100 mL round-bottom flask, separately. The reaction was allowed to proceed at 37 °C for
18 h. After acidification with 5 N H2SO4, the solution was decolorised with a 1 M solution of

NATuRAL PRODuCT RESEARCH
7
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 solution of diazomethane, analysed by GC–MS and found methyl 2-methoxypen-
ta-1,5-dioate (m/z 190) and methyl tridecanoate (m/z 228), respectively.
3.6. Lipoxygenase inhibition assay
The LOX activity was assayed according to the method (Tappel 1953) with slight modifica-
tions. A total volume of 200 µL assay mixture contained 160 µL sodium phosphate buffer
(100 mM, pH 8.0), 10 µL (0.5 mM) of test compound and 20 µL purified lipoxygenase
(EC 1.13.11.12) type-I-B from Glycine max (600 units per well, Sigma-Aldrich Chem. Co., St.
Louis MO, uSA. Cat. No. 9029-60-1). The contents were pre-incubated for 6 min at 25 °C. The
reaction was initiated by addition of 10 µL 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, uSA). The positive and negative controls
were included in the assay. The percentage inhibition (%) was calculated by formula:
Inhibition (%) = (Control − Test) × 100
Control is the total enzyme activity without inhibitor and test is the activity of the test com-
pound. IC50 values (concentration at which there is 50% enzyme inhibition) were determined
after suitable dilutions of the compounds and results computed using EZ-Fit Enzyme kinetics
software (Perrella Scientific Inc., Amherst, uSA).
3.7. Cholinesterase inhibition assay
Electric eel (Electrophorus electricus) AChE (EC 3.1.1.7, type VI-S), equine-serum BChE
(EC 3.1.1.8) inhibiting activities were measured by the method (Ellman et al. 1961). All
chemicals were purchased from Sigma-Aldrich Co., uSA. Total volume of the reaction
mixture 100 µL contained 60 µL KH2PO4 buffer (100 mM, pH 7.7). Ten µL test compound
(0.5 mM per well) was added, followed by the addition of 10 µL enzyme (0.05 units AChE
& 0.5 units BChE). The contents were mixed, pre-incubated for 10 min at 37 °C and pre-
read at 405 nm. The reaction was initiated by the addition of 10 µL of 0.5 mM per well
of respective substrate, followed by the addition of 10 µL DTNB (0.5 mM per well). After
15 min, absorbance was measured at 405 nm. Data were computed as described above
for lipoxygenase assay.
4. Conclusion
Seven compounds were isolated from the whole plant material of C. oxyacantha, including
two new sphingolipids named as oxyacanthins A, B (1, 2). Both the compounds 1 and 2
showed inhibitory potential against enzyme lipoxygenase with IC50 values 83.3 1.3 and
245.7 1.1 µM. However, compound 2 showed inhibitions against enzymes AChE and BChE
with IC50 values 65.3 0.1 and 93.6 0.1 µM, respectively.
Acknowledgements
The authors are thankful to Alaxander von Humboldt (AvH) Foundation, Germany, and Third World
Academy of Science (TWAS), Italy, for providing some financial support.

8
M. DILSHAD ET AL.
References
Ahmad M, Waheed I, Khalil-ur-Rehman M, Niaz u, Saeed-ul-Hassan S. 2007–2010. A review on Carthamus
oxycantha. Pak J Pharm. 20–23:37–41.
Ahmad SS, Wahid A, Bukhsh E, Ahmad S, Kakar SR. 2009. Antihyperlidemic properties of Carthamus
oxyacantha. Pak J Sci. 61:116–121.
Ahmed E, Malik A, Afza N, Riaz N, Anis I, Sharif A, Farheen S, Arif Lodhi MA, Iqbal Choudhary MI. 2007.
Chymotrypsin inhibitory constituents from Haloxylon recurvum. Nat Prod Res. 21:69–75.
Arpornsuwan T, Changsri K, Roytrakul S, Punjanon T. 2010. The effects of the extracts from Carthamus
tinctorius L. on gene expression related to cholesterol metabolism in rats. Songklanakarin J Sci
Tech. 32:129–136.
Arpornsuwan T, Petvises S, Thimuam A, Boondech A, Roytrakul S. 2012. Effects of Carthamus tinctorius
L. solvent extracts on anti-proliferation of human colon cancer (SW 620 cell line) via apoptosis and
the growth promotion of lymphocytes. Songklanakarin J Sci Tech. 34:45–52.
Bernard AA, Eokes L. 1977. Electron impact induced fragmentation of cholesterol and related C-5
unsaturated steroids. J Org Chem. 42:725–732.
Bocheva A, Mikhova B, Taskova R, Mitova M, Duddeck H. 2003. Antiinflammatory and analgesic effects
of Carthamus lanatus aerial parts. Fitoterapia. 74:559–563.
Dongfack MDJ, Lallemand MC, Kuete V, Mbazoa CD, Wansi JD, Trinh-van-Dufat H, Michel S, Wandji J.
2012. A new sphingolipid and furanocoumarins with antimicrobial activity from Ficus exasperata.
Chem Pharm Bull. 60:1072–1075.
Ellman GL, Courtney KD, Andres V, Featherstone RM. 1961. A new and rapid colorimetric determination
of acetylcholinesterase activity. Biochem Pharmacol. 7:88–95.
Gao J-M, Zhang A-L, Chen H, Liu J-K. 2004. Molecular species of ceramides from the ascomycete truffle
Tuber indicum. Chem Phys Lipids. 131:205–213.
Hacioglu BT, Arslan Y, Subasi I, Katar D, Bulbul AS, Ceter T. 2012. Achene morphology of Turkish
Carthamus species. Aust J Crop Sci. 6:1260–1264.
Han S-Y, Li H-X, Ma X, Zhang K, Ma Z-Z, Tu P-F. 2009. Protective effects of purified safflower extract on
myocardial ischemia in vivo and in vitro. Phytomedicine. 16:694–702.
Hannun YA, Obeid LM. 2008. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat
Rev Mol Cell Biol. 9:139–150.
Holland HL, Diakow PRP, Taylor GJ. 1978. 13 C nuclear magnetic resonance spectra of some C-19-hydroxy,
C-5,6 epoxy, C-24 ethyl, and C-19-norsteroids. Can J Chem. 56:3121–3127.
Jun MS, Ha YM, Kim HS, Jang HJ, Kim YM, Lee YS, Kim HJ, Seo HG, Lee JH, Lee SH, et al. 2011. Anti-
inflammatory action of methanol extract of Carthamus tinctorius involves in heme oxygenase-1
induction. J Ethnopharmacol. 133:524–530.
Kang J, Huo CH, Li Z, Li ZP. 2007. New ceramides from the flower of Albizia julibrissin. Chin Chem Lett.
18:181–184.
Khan SW, Khatoon S. 2008. Ethnobotanical studies on some useful herbs of Haramosh and Bugrote
valleys in Gilgit, northern areas of Pakistan. Pak J Bot. 40:43–58.
Kwon HC, Lee KC, Cho OR, Jung IY, Cho SY, Kim SY, Lee KR. 2003. Sphingolipids from bombycis corpus
101A and their neurotrophic effects. J Nat Prod. 66:466–469.
Li H-X, Han S-Y, Wang X-W, Ma X, Zhang K, Wang L, Ma Z-Z, Tu P-F. 2009. Effect of the carthamins yellow
from Carthamus tinctorius L. on hemorheological disorders of blood stasis in rats. Food ChemToxicol.
47:1797–1802.
Matthaus B, Özcan MM, Al-Juhaimi FY. 2015. Fatty acid composition and tocopherol profiles of safflower
( Carthamus tinctorius L.) seed oils. Nat Prod Res. 29:193–196.
Muralidhar P, Kumar MM, Krishna N, Rao CB, Rao DV. 2005. New sphingolipids and a sterol from a
Lobophytum species of the Indian Ocean. Chem Pharm Bull. 53:168–171.
Riaz N, Nawaz SA, Mukhtar N, Malik A, Afza N, Ali S, ullah S, Muhammad P, Choudhary MI. 2007. Isolation
and enzyme-inhibition studies of the chemical constituents from Ajuga bracteosa. Chem Biodiv.
4:72–83.
Riaz N, Parveen S, Saleem M, Ali MS, Malik A, Ashraf M, Afzal I, Jabbar A. 2012. Lipoxygenase inhibitory
sphingolipids from Launaea nudicaulis. J Asian Nat Prod Res. 14:545–554.

NATuRAL PRODuCT RESEARCH
9
Rubinstein I, Goad LJ, Clague ADH, Mulheirn LJ. 1976. The 220 MHz NMR spectra of phytosterols.
Phytochemistry. 15:195–200.
Safder M, Riaz N, Imran M, Nawaz H, Malik A, Jabbar A. 2009. Phytochemical studies on Asphodelus
tenuifolius. J Chem Soc Pak. 21:122–125.
Shibuya H, Kawashima K, Sakagami M, Kawanishi H, Shimomura M, Ohashi K, Kitagawa I. 1990.
Sphingolipids and glycerolipids. I. Chemical structures and ionophoretic activities of soya-
cerebrosides I and II from soybean. Chem Pharm Bull. 38:2933–2938.
Talebi M, Mokhtari N, Rahimmalek M, Sahhafi SR. 2012. Molecular characterization of Carthamus
tinctorius and C. oxyacanthus germplasm using sequence related amplified polymorphism (SRAP)
markers. Plant Omics J. 5:136–142.
Tappel AL. 1953. The mechanism of the oxidation of unsaturated fatty acids catalyzed by hematin
compounds. Arch Biochem Biophys. 44:378–395.
Taskova R, Mitova M, Najdenski H, Tzvetkova I, Duddeck H. 2002. Antimicrobial activity and cytotoxicity
of Carthamus lanatus. Fitoterapia. 73:540–543.
Topashka-Ancheva MN, Taskova RM, Handjieva NV. 2006. Mitogenic effect of Carthamus lanatus
extracts, fractions and constituents. Fitoterapia. 77:608–610.
Wang M, Li J, Ho GS, Peng X, Ho C-T. 1998. Isolation and identification of antioxidative flavonoid
glycosides from thyme (Thymus Vulgaris L.). J Food Lipids. 5:313–321.