Ascorbic acid 6-palmitate: A potent inhibitor of human and soybean lipoxygenase-dependent lipid peroxidation

Article abstract of DOI:10.1111/jphp.12200

Objectives Lipoxygenases (LOX) are the key enzymes involved in the biosynthesis of leukotrienes and reactive oxygen species, which are implicated in pathophysiology of inflammatory disorders. This study was conducted to evaluate the inhibitory effect of water-soluble antioxidant ascorbic acid and its lipophilic derivative, ascorbic acid 6-palmitate (Vcpal) on polymorphonuclear lymphocyte 5-LOX and soybean 15-LOX (sLOX) in vitro. Methods LOX activity was determined by measuring the end products, 5-hydroperoxy eicosatetraenoic acid (5-HETE) and lipid hydroperoxides, by spectrophotometric and high performance liquid chromatography methods. The substrate-dependent enzyme kinetics and docking studies were carried out to understand the nature of inhibition. Key findings Vcpal potently inhibited 5-LOX when compared with its inhibitory effect on sLOX (IC50; 2.5 and 10.3μm respectively, P= 0.003). Further, Vcpal inhibited 5-LOX more strongly than the known synthetic drugs: phenidone and nordihydroguaiaretic acid (P= 0.0007). Enzyme kinetic studies demonstrated Vcpal as a non-competitive reversible inhibitor of 5-LOX. In-silico molecular docking revealed high MolDock and Rerank score for Vcpal than ascorbic acid, complementing in-vitro results. Conclusion Both in-vitro and docking studies demonstrated Vcpal but not ascorbic acid as a non-competitive inhibitor of 5-LOX- and sLOX-induced lipid peroxidation, suggesting a key role for lipophilic nature in bringing about inhibition.

Full text of DOI:10.1111/jphp.12200

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Research Paper
Journal of Pharmacy
And Pharmacology
Ascorbic acid 6-palmitate: a potent inhibitor of human and
soybean lipoxygenase-dependent lipid peroxidation
Riyaz Mohameda, Shaista Tarannuma, Manjunath Yariswamya, Hamse K. Vivekb,
Jalahalli M. Siddeshaa, Nataraju Angaswamya and Bannikuppe S. Vishwanatha
aDepartment of Studies in Biochemistry, University of Mysore and bSri Jayachamarajendra College of Engineering, JSS Technical campus, Mysore,
India
Keywords
ascorbic acid 6-palmitate; human; lipid
Abstract
Objectives Lipoxygenases (LOX) are the key enzymes involved in the biosynthesis
of leukotrienes and reactive oxygen species, which are implicated in pathophysiol-
ogy of inflammatory disorders. This study was conducted to evaluate the inhibi-
tory effect of water-soluble antioxidant ascorbic acid and its lipophilic derivative,
ascorbic acid 6-palmitate (Vcpal) on polymorphonuclear lymphocyte 5-LOX and
soybean 15-LOX (sLOX) in vitro.
peroxidation; lipoxygenase; soybean
Correspondence
Bannikuppe S. Vishwanath, Department of
Studies in Biochemistry, University of Mysore,
Manasagangothri, Mysore 570006,
Karnataka, India.
Methods LOX activity was determined by measuring the end products,
5-hydroperoxy eicosatetraenoic acid (5-HETE) and lipid hydroperoxides, by
spectrophotometric and high performance liquid chromatography methods. The
substrate-dependent enzyme kinetics and docking studies were carried out to
understand the nature of inhibition.
E-mail: vishmy@yahoo.co.uk
Received April 2, 2013
Accepted November 16, 2013
doi: 10.1111/jphp.12200
Key findings Vcpal potently inhibited 5-LOX when compared with its inhibitory
effect on sLOX (IC50; 2.5 and 10.3 μm respectively, P = 0.003). Further, Vcpal
inhibited 5-LOX more strongly than the known synthetic drugs: phenidone and
nordihydroguaiaretic acid (P = 0.0007). Enzyme kinetic studies demonstrated
Vcpal as a non-competitive reversible inhibitor of 5-LOX. In-silico molecular
docking revealed high MolDock and Rerank score for Vcpal than ascorbic acid,
complementing in-vitro results.
Conclusion Both in-vitro and docking studies demonstrated Vcpal but not
ascorbic acid as a non-competitive inhibitor of 5-LOX- and sLOX-induced lipid
peroxidation, suggesting a key role for lipophilic nature in bringing about
inhibition.
Introduction
Lipoxygenases (LOXs) are a family of non-heme, iron-
containing enzymes widely distributed among animals and
plants. In mammals, three major types of LOX have been
identified, namely 5-, 12- and 15-LOX, which insert
dioxygen at the C5, C12 and C15 positions of arachidonic
acid (AA), respectively.[1] 5-LOX, a key enzyme in the
leukotriene (LTs) biosynthetic pathway, is responsible for
the synthesis of LTB4, LTC4, LTD4, LTE4 and
5-hydroperoxy eicosatetraenoic acid (5-HETE) from AA.[2]
These LTs formed due to 5-LOX activity are powerful
mediators of inflammation and regulators of the immune
system.[3]
Furthermore, the distribution of 5-LOX is essentially
restricted to polymorphonuclear leukocytes (PMNLs),
monocytes, macrophages, mast cells and B-lymphocytes.
Therefore, LTs-mediated inflammation is most commonly
associated with diseases such as asthma, atherosclerosis,
chronic obstructive pulmonary disease, rheumatoid arthri-
tis, cancer, liver fibrosis and Alzheimer’s disease.[4–7] Addi-
tionally, 15-LOX is of particular interest since it can also
oxidize the esterified fatty acids in biological membranes
and lipoproteins, forming 15-hydroperoxy-eicosatetraenoic
acid (15-HPETE) from AA and 13-hydroperoxy-
octadecadienoic acid (13-HPODE) from linoleic acid.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778
769

Ascorbic acid 6-palmitate and lipid peroxidation
Riyaz Mohamed et al.
Mammalian 5-LOX and 15-LOX have been implicated in
other specific oxidative reactions relevant in the pathogen-
esis of atherosclerosis.[8,9] Alternatively, soybean lipoxy-
genase (sLOX) is a plant-derived 15-LOX that efficiently
catalyses the oxidation of linoleic acid to 13-HPODE
similar to mammalian 15-LOX. Based on the structural and
functional similarities with mammalian LOXs, sLOX is
commonly used for both mechanistic and inhibitory
studies, and is widely accepted as a model for LOXs from
other sources.[10,11]
During the biosynthesis of inflammatory mediators such
as LTs, various reactive oxygen species (ROS) are generated
as by-products. These free radicals augment the develop-
ment of inflammatory reactions, along with production of
pro-inflammatory LTs. The generated ROS also bring about
lipid peroxidation, producing lipid peroxides and other
cytotoxic lipid derivatives.[12] Thus, the lipid peroxides that
are formed are highly reactive molecules, capable of altering
the membrane fluidity, ion permeability, surface charge,
membrane enzyme activity, cell signalling and can damage
DNA, all of which alter the cellular functions. Such changes
are assumed to be causative factors for ageing and are asso-
ciated with several degenerative disorders.[2,12,13] In such
conditions, the scavenging of free radicals, along with the
inhibition of inflammatory LTs production through the
inhibition of LOX pathways by single antioxidant molecule,
will have a prospective therapeutic role in the treatment of a
variety of inflammatory and allergic diseases.
enzymes involved in LTs-mediated inflammatory disease
conditions. Further, docking studies were performed to
determine the possible enzyme inhibitor interaction.
Materials and Methods
Chemicals
All the chemicals used were analytical grade. Ascorbic acid,
ascorbic acid 6-palmitate (structure shown in Figure 1),
adenosine triphosphate (ATP), dithiothreitol (DTT), AA,
sLOX, linoleic acid, phenidone and nordihydroguaiaretic
acid (NDGA) were obtained from Sigma Chemicals Co.
(Saint Louis, MO, USA). Fatty acid was purchased from
Sisco Research Laboratories Pvt. Ltd. (Mumbai, India). Sol-
vents used for high performance liquid chromatography
(HPLC) analysis were HPLC grade obtained from Ranbaxy
Fine Chemicals Ltd (New Delhi, India).
Isolation of 5-lipoxygenase enzyme
from polymorphonuclear leukocytes of
human blood
Venous blood from healthy individuals who were not under
any medication was collected into ethylene diamine
tetraacetic acid (EDTA)-containing tubes. The protocols
were reviewed by the Institutional Human Ethical Commit-
tee, University of Mysore (Sanction order No. IHEC-UOM
No.40/Ph. D/2009-10). PMNL cells were isolated from
Many natural and synthetic compounds with redox
and non-redox potential are known to inhibit LOX
enzymes.[14–16] Ascorbic acid is one of the most abundant,
naturally occurring, water-soluble antioxidant that has been
broadly studied for the treatment of many inflammatory
disorders due to its effective antioxidant property, but its
anti-inflammatory property is still controversial.[17–19]
Further, ascorbic acid 6-palmitate (Vcpal), a synthetic
hydrophobic ascorbic acid derivative with strong antioxi-
dant property, is extensively studied for its hydrophobic
property.[20–23] Since Vcpal is not naturally found in food
sources, synthetic VC6P is used as a source of vitamin C and
as an antioxidant food additive. In addition, it is also used
as an antioxidant in processed foods like breads and
peanuts as well as it has been used to increase the shelf life
of vegetable oils and potato chips.[24–26] Previous studies
have revealed that Vcpal prevents the peroxidation of low-
density lipoprotein and protect human erythrocytes from
oxidative damage.[27]
(a)
(b)
OH
O
O
O
H
Interestingly, despite seemingly extensive characterization
of the antioxidant effects of Vcpal, it is still unknown
whether this compound could inhibit human 5-LOX and
soybean 15-LOX. Therefore, we examined the inhibitory
potential of ascorbic acid and its synthetic lipophilic deriva-
tive Vcpal using human 5-LOX and soybean LOX, the key
HO
OH
H3C
Figure 1 Chemical structure of ascorbic acid (a) and ascorbic acid
6-palmitate (Vcpal) (b).
770
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Riyaz Mohamed et al.
Ascorbic acid 6-palmitate and lipid peroxidation
blood by Ficoll-Histopaque density gradient and hypotonic
lysis of erythrocytes.[28] All the procedures were performed
at 4°C. PMNL cells were re-suspended in the phosphate
buffer saline and sonicated for 20–30 s at 20 kHz to release
the cytosolic 5-LOX enzyme into solution. This solution
was centrifuged at 100 000g for 30 min at 4°C, and the
supernatant obtained was directly used as enzyme source.
Protein concentration was estimated by Lowry’s method
using fat-free bovine serum albumin as standard.
as described above. The ICmin, IC50 and IC100 concen-
trations were determined by Boltzmann dose response
analysis.
Enzyme kinetic studies on inhibition of
5-lipoxygenase with Vcpal
The enzyme kinetics on inhibition of 5-LOX was studied
using different concentrations of substrate AA (50 and
200 μm) vs IC25, IC50 and IC75 inhibitory concentrations
of inhibitor. Double reciprocal plots of enzyme kinetics
were constructed according to Lineweaver and Burk.[31]
5-lipoxygenase enzyme assay
The enzyme assay was performed according to the method
published by Aharony and Stein.[29] The standard reaction
mixture for the 5-LOX assay contained 100 mM phosphate
buffer pH 7.4, 50 μm of DDT, 200 μm of ATP, 300 μm of
CaCl2, 150 μm of AA and 5 μg of protein. Enzymatic reac-
tions were carried out at room temperature. 5-LOX activity
was measured as the amount of 5-HETE formed at 234 nm
using Shimadzu 160A spectrophotometer (Tokyo, Japan).
The molar extinction coefficient of 28 000/M/cm was used
to calculate the specific activity of enzyme. The enzyme
activity was expressed as μmol of 5-HETE formed/min/mg
protein.
HPLC separation of 5-lipoxygenase product
5-HETE and its inhibition by Vcpal
5-LOX product 5-HETE was resolved by HPLC method on
a Luna straight phase silica column (2.5 mm × 25 cm).
Enzyme assay was carried at room temperature as described
above, in the presence and absence of Vcpal. The reaction
mixture contained 100 mM phosphate buffer pH 7.4, 50 μm
of DTT, 200 μm of ATP, 300 μm of CaCl2, 150 μm of AA and
5 μg of protein. The reaction was stopped with the addition
of 0.5 ml of 6N HCl at the end of 5 min. The 5-LOX
product, namely 5-HETE, was extracted twice using organic
solvent mixture of hexane and diethyl ether (1 : 1). The
organic layers were pooled, dried under nitrogen and finally
dissolved in 50 μl of mobile phase. The sample (10 μl) was
injected on a straight phase silica column, and 5-HETE
Soybean lipoxygenase enzyme assay
Enzymatic lipid peroxidation was measured spectro-
photometrically following an increase in absorbance of lipid
hydroperoxides formation at 234 nm.[30] The reaction
medium contained 20 μm linoleic acid and 4 μg sLOX
enzyme in 50 mM Tris buffer pH 7.4. The increase in
absorbance at 234 nm was monitored by a Shimadzu UV
160A spectrophotometer. The molar extinction coefficient
of 25 mM/cm was used to calculate the specific activity of
enzyme. The enzyme activity was expressed as μmol of
hydroperoxide formed/min/mg protein under the assay
conditions.
was resolved using
a
mobile phase of hexane :
isopropanol : acetic acid (100 : 2 : 0.1) with a flow rate of
0.5 ml/min at 234 nm. The product 5-HETE formed was
confirmed using an authentic 5-HETE standard obtained
from Cayman Chemicals (Ann Arbor, MI, USA). The
percent inhibition of 5-HETE levels was calculated from
peak area of the control and inhibitor-treated sample.
Molecular docking
Molecular docking was carried out using Molegro Virtual
Docker (MVD).[32] MVD is based on differential evaluation
algorithm; docking scoring function, E score and the solu-
tion of algorithm, taking into account the sum of the inter-
molecular interaction energy between the ligand and the
protein (Einter), and the intramolecular energy of the ligand
(Eintra). The structures of the ligands were constructed and
optimized using the Dundee PRODRG2 Server[33] and saved
in the MOL format. The crystal structure of soybean
lipoxygenase-3 (protein data base (PDB) code: 1NO3) was
obtained from the PDB database,[34] and it shares 63%
sequence identity with human 5-LOX, which was retrieved
using an automated docking MVD software platform,
that is based on guided differential evolution and a force
field-based screening function.[32] For the convenience of
Inhibitory studies with Vcpal on
polymorphonuclear leukocyte
5-lipoxygenase and soybean
15-lipoxygenase
Vcpal, phenidone, palmitate and NDGA were dissolved in
dimethyl sulfoxide (DMSO) at 2.0 mM concentration and
pre-incubated with enzymes (human 5-LOX and sLOX) at
different concentrations for 2.0 min, and then the reaction
was initiated with addition of AA and linoleate separately.
Controls with DMSO were maintained to check the effect of
DMSO on lipid peroxidation reaction. The activity of
5-LOX and sLOX were measured as 5-HETE and lipid
hydroperoxides formed at 234 nm spectrophotometrically,
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778
771

Ascorbic acid 6-palmitate and lipid peroxidation
Riyaz Mohamed et al.
docking, all water molecules and the inhibitor were
excluded, whereas ferrous atom was retained in the active
site, fixed to their crystal positions, throughout the docking
process, necessary bonds, bond orders and hybridization,
explicit hydrogens and protein charges were assigned and
flexible torsion of ligands were detected. The Molegro
docking wizard was used to simulate in-silico docking of
ligands into sLOX using a MolDock scoring function of grid
resolution of 0.30 Å, and search algorithm MolDock SE was
used and a minimum of 10 independent runs per ligand
were performed for better docking result. A value of popu-
lation size and maximum interactions 100 and 10 000,
respectively, were used for each run, and five best poses were
retained for each ligand. Molecular visualization was done
using Molegro Molecular Viewer 2008 version 1.2.0 (CLC
bio, Cambridge, MA, USA).
5-HETE is eluted as a single distinct peak at 7.88 min. Incu-
bation of 5-LOX with different concentrations of Vcpal
resulted in dose-dependent decrease in the levels of
5-HETE, as shown in Table 2. At 3.5 μm concentration,
Vcpal exhibited 72 5.0% inhibition.
Further, enzyme kinetic studies were carried out to
understand the mechanism of inhibition of PMNLs 5-LOX
by Vcpal. Substrate-dependent inhibitory activity was
evaluated by varying concentrations of AA (50–200 μm)
and different concentrations of Vcpal (IC25, IC50 and
IC75). A double reciprocal plot of substrate vs enzyme
velocity in the presence of Vcpal is presented in Figure 4.
Vcpal treatment at 0, 1.6, 2.5 and 3.6 μm decreased the
maximum velocity (Vmax) of the enzyme activity from
3.45 to 2.70, 2.15 and 1.65 μmol of 5-HETE/min/mg of
protein, respectively, without much change in the Km value
112, 109, 114 and 112 μm (Table 3). The inhibitory constant
Statistical analysis
The data are presented as specific activity of 5-LOX enzyme
in the presence of different concentrations of inhibitors.
The ICmin, IC25, IC50, IC75 and IC100 values were calcu-
lated by Boltzmann dose response analysis. Statistical differ-
ences were calculated by Duncan’s multiple range tests at
P < 0.05 using the SPSS statistical software (IBM Corp.,
Armonk, NY, USA). The results are presented as mean
standard deviation of at least four determinations.
1.6
1.2
0.8
0.4
0
Results
The inhibitory effect of Vcpal on human PMNLs 5-LOX
activity was evaluated by measuring the formation of
5-LOX product 5-HETE by spectrophotometric and
HPLC methods. Vcpal inhibited PMNLs 5-LOX in a
concentration-dependent manner, with ICmin, IC50 and
IC100 value of 0.4 μm, 2.5 μm and 4.8 μm concentrations,
respectively (Figure 2). The parent compound ascorbic acid
failed to inhibit human PMNLs 5-LOX, whereas the inhibi-
tory effect of palmitate was not significant when compared
with Vcpal, even at higher concentrations tested. The IC50
value of Vcpal for inhibition of 5-LOX activity in compari-
son with synthetic 5-LOX inhibitors is presented in Table 1.
Based on the IC50 values, the inhibitory effect of Vcpal on
human 5-LOX was fivefold more potent compared with its
effect on sLOX (P = 0.003). Further, Vcpal inhibited human
5-LOX 9.6 and 11.2-fold more strongly compared with
known synthetic standard drugs phenidone and NDGA
(P = 0.0007). This data demonstrate that 5-LOX has more
affinity for Vcpal when compared with soybean 15-LOX
enzyme. Furthermore, this data support Vcpal as a strong
inhibitor of 5-LOX at physiological concentrations.
0
1
2
3
4
5
Ascorbic acid-6 hexadeconoate (µM)
Figure 2 Dose-dependent inhibition of human polymorphonuclear
leukocytes 5-lipoxygenase enzyme by Vcpal. Vcpal was incubated with
5-lipoxygenase enzyme isolated from human polymorphonuclear leu-
kocytes for 2 min before starting the reaction by adding substrate. The
enzyme activity was followed spectrophotometrically at 234 nm. The
data are represented as mean standard deviation for four individual
experiments.
Table 1 IC50 values of Vcpal for inhibition of human 5-lipoxygenase
and soybean lipoxygenase activity in comparison with standard syn-
thetic 5-lipoxygenase inhibitors
Compounds
Enzyme source
IC50 valuea (μM)
Ascorbic acid
Vcpal
5-LOX/soybean LOX
5-LOX
00
2.5
10.3
24
Vcpal
Soybean LOX
5-LOX
Phenidone
NDGA
5-LOX
28
NDGA, nordihydroguaiaretic acid. aIC50 value is defined as the amount
of inhibitor (μM) required to inhibit 50% of enzyme activity in the given
reaction mixture. The linear regression analysis of the linear portion of
the dose-dependent inhibition curve of each activity was used for the
calculation of IC50.
The inhibitory action of Vcpal on 5-LOX was further
confirmed by HPLC method (Figure 3). The 5-LOX product
5-HETE was resolved on silica column by HPLC at 234 nm.
772
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Riyaz Mohamed et al.
Ascorbic acid 6-palmitate and lipid peroxidation
mAU
234 nm 4 nm (1.00)
mAU
100 234 nm 4 nm (1.00)
125
100
75
(a)
(b)
75
Vcpal-1.5 µM
50
25
50
25
0
0
−25
−50
−25
−50
0.0
0.0
2.5
5.0
7.5 10.0 12.5 15.0 17.5 min
min
2.5
5.0
7.5
10.0 12.5 15.0
mAU
mAU
234 nm 4 nm (1.00)
234 nm 4 nm (1.00)
100
100
75
50
25
0
Vcpal-3.5 µM
(d)
(c)
Vcpal-3.0 µM
75
50
25
0
−25
−25
−50
−50
min
0.0
2.5
5.0
7.5
10.0 12.5 15.0
min
0.0
2.5
5.0
7.5
10.0
12.5 15.0
Figure 3 HPLC chromatograph of HETE resolved on silica column. A dose-dependent reduction in 5-HETE content was observed in the presence of
Vcpal (b–d).
Table 2 Inhibitory effect of Vcpal on 5-lipoxygenase product 5-HETE
content using HPLC method
2
Control
1.6 µM
2.5 µM
3.6 µM
Vcpal concentration (μM)
% inhibition compared with control
1.6
0.0
1.5
3.0
3.5
0.0
1.2
0.8
0.4
0
42.0 3.53
70.6 6.70
73.0 3.35
Ki for Vcpal was found to be 0.37 μm. The enzyme kinetic
data demonstrated that Vcpal is a non-competitive revers-
ible inhibitor of human 5-LOX enzyme.
–0.01
–0.005
0
0.005
1/[S]
0.001
0.0015
0.02
Lipid peroxidation consists of a radical-initiated reaction
and can serve as suitable system for evaluating the antioxi-
dant property. A number of LOX inhibitors are considered
as antioxidant and are good inhibitors of lipid peroxidation.
We further determined the effect of Vcpal and ascorbic acid
on soybean 15-LOX-induced lipid peroxidation. Vcpal but
not ascorbic acid significantly inhibited sLOX-dependent
linoleic acid peroxidation by reducing hydroperoxide pro-
duction with an ICmin, IC50 and IC100 value of 0.9 μm,
10.3 μm and 27 μm concentrations, respectively (Figure 5),
Figure 4 A double reciprocal plot for substrate-dependent inhibition
kinetics of human polymorphonuclear leukocytes 5-lipoxygenase
enzyme activity by Vcpal. 5-lipoxygenase enzyme was incubated with
different concentrations of the inhibitor Vcpal in the presence of
various concentrations of the substrate arachidonic acid. The changes
in the Km values were not observed in the presence of different con-
centration of Vcpal.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778
773

Ascorbic acid 6-palmitate and lipid peroxidation
Riyaz Mohamed et al.
Table 3 Effect of Vcpal on Vmax and Km valves of human
polymorphonuclear leukocyte 5-lipoxygenase enzyme
reveal an efficient MolDock score and Rerank score when
compared with standard inhibitors (Table 4). These docking
scoring functions demonstrate the lead molecule Vcpal as a
good inhibitor of LOX enzymes, whereas vitamin C fits into
the hydrophilic core of the enzyme to form hydrogen bonds
with surrounding amino acid residues. Figure 6b depicts the
binding mode of ascorbic acid with sLOX. The oxygen
atoms of ascorbic acid ring form hydrogen bonds with
Val589 (2.63 Å), Asp592 (3.18 Å) and Trp593 (3.28 Å),
Asn521 (3.31 Å, 2.78 Å and 2.73 Å), Gln598 (3.17 Å and
2.75 Å), Val594 (2.97 Å), Gln598 (2.86 Å and 2.75 Å), and
Asp597 (2.78 Å). As shown in Figure 6c, the known stand-
ard inhibitor phenidone binds to the hydrophilic core of the
enzyme, and forms hydrogen bonds with Asp375 (3.03 Å)
and Ser517 (2.79 Å). MolDock score and Rerank score of
acorbic acid, Vcpal and phenidone are given in Table 4.
Vcpal
Vmax (μmol of
concentration (μM)
Km (μM)
HETE/min/μg protein)
0.0
1.6
2.5
3.5
112.0
109.0
114.0
112.0
3.45
2.70
2.15
1.65
1.6
1.2
0.8
0.4
0
Discussion
The PMNLs play an important role in the modulation of
inflammatory responses. PMNLs are initially recruited at
the site of injury and secrete elevated levels of leukotrienes,
prostaglandins and ROS, which contribute to inflammatory
pathophysiology.[1,13] Especially, LTs along with ROS acceler-
ate the progression of the diseases by exacerbating the
initial local inflammatory responses that include vasodilata-
tion, local extravasation and leakage of blood vessels, even-
tually leading to tissue damage.[5–7,35] The production and
release of LTs and ROS can be prevented by inhibition of
LOX pathways by antioxidants and their derivatives. Ascor-
bic acid is one such naturally occurring antioxidant that
directly reacts with aqueous peroxyl radicals and also
indirectly co-stimulates the antioxidant properties of fat-
soluble vitamin E.[17,18] Williams et al. demonstrated the
inhibition of degranulation of PMNLs by ascorbic acid,
leading to decreased release of myeloperoxidase, thus
preventing potential tissue damage.[36] The earlier study
also demonstrated the ability of ascorbic acid to inhibit
the conversion of AA to PGF2α in guinea pig uterus.[37]
Further, ascorbic acid also reduced the production of
12-hydoxyeicosatetraenoic acid, the major LOX product
from human corneal tissue[38] confirming its interference in
prostaglandin biosynthesis. Since ascorbic acid rapidly
undergoes oxidation and irreversible degradation in the
body,[37] many researchers reveal that antioxidant activity of
ascorbic acid could be significantly enhanced by introduc-
ing a hydrocarbon tail to make it more stable and lipid-
soluble.[23,39] Additionally, these synthetic derivatives are
extensively studied for its antioxidant, antitumor and anti-
inflammatory activity.[19–23,40–42] Ascorbic acid 6-palmitate
(Vcpal), a synthetic lipophilic amphipathic derivative, is
used as an antioxidant in food additives, vitamins, drug and
dermatological products. The antioxidant potential of Vcpal
0
5
10
15
20
25
30
Ascorbic acid-6 hexadeconoate (µM)
Figure 5 Dose-dependent inhibition of soybean lipoxygenase enzyme
by Vcpal. Vcpal was incubated with soybean lipoxygenase enzyme for
2 min before starting the reaction by adding substrate. The enzyme
activity was followed spectrophotometrically at 234 nm. The data
are represented as mean standard deviation for four individual
experiments.
while ascorbic acid failed to inhibit even at higher concen-
trations tested (Table 1). Comparison of dose linear inhibi-
tion of 5-LOX and 15-LOX by Vcpal elucidate that Vcpal is
fivefold more potent inhibitor of human 5-LOX than
soybean 15-LOX enzyme (Figure 2 and Figure 5).
Further, the docking studies were performed to deter-
mine the possible mechanism of action, as well as the differ-
ences in the specificity of the compound towards LOX
enzymes. As shown in Figure 6a, the interaction between
Vcpal and sLOX reveals a typical inhibitory mechanism for
a potent inhibitor of 5-LOX, wherein its polar head and
long hydrophobic tail play a crucial role in its better binding
for the inhibition. The hydroxyl oxygen atoms present in
the furan ring of Vcpal form hydrogen bonds with Arg386
(3.26 Å), Pro384 (2.75 Å), Asp382 (3.01 Å), Leu380 (2.93 Å)
and Asp382 (2.90 Å). Additionally, the carboxyl oxygen of
fatty acid backbone, which is involved in esterification
linkage with ascorbic acid moiety, forms hydrogen bonds
with Asp378 (3.20 Å and 3.11 Å). The hydrogen bond deter-
mines the best pose orientation for Vcpal in the binding site
and stabilizes the hydrophobic interaction of its hydropho-
bic tail with surrounding amino acid residues Leu399,
Ser387, Arg386, Pro423, Asp431, Val589 and Trp592, which
774
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778

Riyaz Mohamed et al.
Ascorbic acid 6-palmitate and lipid peroxidation
(a)
(b)
(c)
Figure 6 Schematic representation of hydrogen bond and hydrophobic interactions of soybean lipoxygenase with ascorbic acid (a), ascorbic acid-6
palmitate (b) and phenidone (c). Highlight the surrounding residues, electrostatic interaction and hydrogen bond interaction. Ligands are shown in
stick model, and dotted line indicates hydrogen bond.
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778
775

Ascorbic acid 6-palmitate and lipid peroxidation
Riyaz Mohamed et al.
Table 4 Molecular docking results
The inhibitory mechanism of 5-LOX studied by AA- (50–
200 μm) dependent enzyme kinetics revealed that Vcpal
significantly did not change the Km value of 5-LOX
(∼112 3 μm, P = 0.08) but decreased the maximum veloc-
ity (Vmax, 3.45 μmol of 5-HETE to 1.65 μmol, P = 0.04) of
the enzyme, suggesting a competitive inhibition. These
results also demonstrate that Vcpal, in addition to binding
at the active site, may also interact with different sites of the
enzyme. Supporting our observation, similar earlier report
by Raghavenra et al. also demonstrated that eugenol at dif-
ferent concentrations did not change the Km value of
5-LOX but decreased the Vmax.[16] Furthermore, this sug-
gests that antioxidant compounds like Eugenol and Vcpal
can scavenge the lipid peroxyl radicals formed during the
oxidation of AA, and thus block the formation of 5-HETE
as evidenced by the decrease in Vmax during enzyme kinet-
ics experiments (Figure 4).
Plant-derived soybean LOX (sLOX) is structurally and
functionally similar to mammalian LOXs that efficiently
catalyse the oxidation of linoleic acid to 13-HPODE. There-
fore, sLOX is commonly used as a mammalian model mol-
ecule to study the mechanistic and inhibitory properties of
human 12/15LOXs.[14,42] By virtue of their hydrophobic
interaction with catalytic site, many n-alcohols as well as
synthetic hydrophobic thiols inhibit sLOX.[45] Similarly in
our study, Vcpal but not ascorbic acid significantly inhibits
sLOX in a dose-dependent manner, with an IC50 of 13.4 μm
concentration (Table 1, Figure 5). This data strongly
confirm that the hydrophobicity of the inhibitor is essential
for LOX inhibition.
The use of plant LOX to model mammalian LOXs will
prove highly beneficial and supportive in structural charac-
terization, mechanism elucidation and possibly the discov-
ery of novel inhibitors of LOXs.[46] Due to unavailability of
three-dimensional structures of 5-LOX, we used crystal
structure of plant sLOX for our docking studies. Generally,
the enzyme/inhibitor complexes are stabilized almost
entirely by van der Waals, aromatic interactions in the
hydrophobic region and by hydrogen bond in hydrophilic
region.[32] Our docking study validates that Vcpal and ascor-
bic acid bind to the hydrophilic core of the enzyme and
forms a hydrogen bond with surrounding amino acid resi-
dues. Vcpal had maximum interaction with high negative
MolDock and Rerank scores, supporting the observed high
inhibitory potency (2.5 μm for 5-LOX and 13 μm for sLOX),
compared with ascorbic acid and phenidone (Table 4). This
demonstrates the strong and specific binding of Vcpal into
hydrophilic pocket of LOX enzyme.
Compound
MolDock score
Rerank score
Ascorbic acid
Vcpal
–83.20
–137.29
–74.375
–71.86
–101.87
–66.1619
Phenidone
The compounds were ranked on the basis of their MolDock and
Rerank scores. The scores of the individual compounds are the best of
five scores derived from independent docking rounds.
is attributed to the inhibition of glutathione s-transferase
enzyme and neutralization of phospholipase A2-induced
local toxicity.[43] However, the detailed mechanism and its
interaction with human 5-LOX and soybean LOX are yet
unknown.
In this study, we investigated the effect of ascorbic acid
and Vcpal on inhibition of PMNLs 5-LOX and soybean
LOX activity. Vcpal significantly inhibited PMNLs 5-LOX in
a concentration-dependent manner with IC50 value of
2.5 μm and inhibitory constant (Ki) of 0.37 μm (Figure 2).
In contrast, ascorbic acid did not inhibit the LOX enzymes.
Further, the extent of 5-LOX inhibition by Vcpal was found
to be tenfold higher than the standard synthetic inhibitors
phenidone and NDGA (P < 0.001) (Table 1). The inhibition
was further confirmed using the HPLC method, wherein
incubation of 5-LOX with various concentrations of Vcpal
resulted in dose-dependent decrease in the end product
5-HETE (73% inhibition at 3.5 μm (Figure 3 and Table 2)).
Consistent with the above results, ascorbic acid did not
inhibit the formation of 5-HETE. The above two results
represent Vcpal as a potent candidate for therapeutic use in
inflammation, with higher efficiency over the parent com-
pound, ascorbic acid.
Ascorbic acid is a weak inhibitor of hyaluronidase and
myeloperoxidases.[23,36] Report by Botzki demonstrated that
Vcpal is a strong inhibitor of hyaluronidase activity com-
pared with the parent compound, ascorbic acid.[23] The long
alkyl chain of Vcpal appeared to interact with an extended,
hydrophobic channel formed by mostly conserved amino
acids of hyaluronidase enzyme.[23] The 5-LOX enzyme with
hydrophobic channels or cavities helps in the conduction of
electrons from substrate to the active site iron atom and
allows a proper interaction with other hydrophobic com-
pounds. This interaction is critical for the inhibition of LOX
enzyme by hydrophobic compounds.[44] Our current obser-
vation further confirms that long hydrophobic alkyl chain
of Vcpal is significant for the improved binding affinity of
this molecule with LOX. These results are also in agreement
with the observation made by Clapp et al., where hydropho-
bic thiols with enhanced hydrophobicity showed more
effective inhibition of LOX activity.[45] Therefore, we infer
that the side chain of Vcpal with additional hydrophobic
residues interacts with the 5-LOX enzyme and enhances the
inhibition of Vcpal compared with ascorbic acid.
Conclusion
This study demonstrates that Vcpal but not the parent
compound L-ascorbic acid as a non-competitive inhibitor
776
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778

Riyaz Mohamed et al.
Ascorbic acid 6-palmitate and lipid peroxidation
of human PMNLs 5-LOX and sLOX induced lipid
peroxidation. The docking studies correlate with observed
in-vitro activity. The data suggest that the lipophilic nature
of the compound plays a critical role in bring about LOX
inhibition.
Acknowledgements and funding
We thank University of Mysore, Mysore for providing facil-
ities to carry out this project. RM, ST and JMS acknowledge
the Council of Scientific and Industrial Research (CSIR),
New Delhi, India for CSIR-SRF.
Declarations
Conflict of interest
The Author(s) declare(s) that they have no conflicts of
interest to disclose.
10. Clemens F et al. The inactivation of 19. Mandl J et al. Vitamin C: update on
References
lipoxygenase-1 from soybeans by
amidrazones. Biochim Biophys Acta
2001; 1549: 88–98.
physiology and pharmacology. Br J
Pharmacol 2009; 157: 1097–1110.
1. Kim C et al. Cytosolic phospholipase
A(2), lipoxygenase metabolites, and
20. Weber V et al. Novel 4,5-diaryl-
reactive oxygen species. BMB Rep 11. Boyington JC et al. The three-
2008; 41: 555–559. dimensional structure of an
2. Radmark et al. 5-Lipoxygenase:
regulation of expression and enzyme
3-hydroxy-2(5H)-furanones as anti-
oxidants and anti-inflammatory agents.
Bioorg Med Chem 2002; 10: 1647–
1658.
O
arachidonic acid 15-lipoxygenase.
Science 1993; 260: 1482–1486.
activity. Trends Biochem Sci 2007; 32: 12. Muralikrishna Adibhatla R, Hatcher 21. Mohamed R et al. Chemical modifica-
332–341.
JF. Phospholipase A2, reactive oxygen
species, and lipid peroxidation in cer-
ebral ischemia. Free Radic Biol Med
2006; 40: 376–387.
tion of ascorbic acid and evaluation of
its lipophilic derivatives as inhibitors
of secretory phospholipase A2 with
anti-inflammatory activity. Mol Cell
Biochem 2010; 345: 69–76.
3. Werz O. Inhibition of 5-lipoxygenase
product synthesis by natural com-
pounds of plant origin. Planta Med
2007; 73: 1331–1357.
13. Nanda BL et al. PLA2 mediated
4. Werz O, Steinhilber D. Therapeutic
options for 5-lipoxygenase inhibitors.
Pharmacol Ther 2006; 112: 701–718.
5. Lotzer K et al. The 5-lipoxygenase
pathway in arterial wall biology and
atherosclerosis. Biochim Biophys Acta
2005; 1736: 30–37.
arachidonate free radicals: PLA2 22. Raic-Malic S. Synthesis and antitumor
inhibition and neutralization of free
radicals by anti-oxidants – a new
role as anti-inflammatory molecule.
Curr Top Med Chem 2007; 7: 765–
777.
activities of novel pyrimidine deriva-
tives of 2,3-O,O-dibenzyl-6-deoxy-L-
ascorbic acid and 4,5-didehydro-5,6-
dideoxy-L-ascorbic acid. J Med Chem
2000; 43: 4806–4811.
14. Lapenna D et al. Dihydrolipoic acid 23. Botzki A. L-Ascorbic acid 6-
6. Pratico D et al. 12/15-lipoxygenase is
increased in Alzheimer’s disease: pos-
sible involvement in brain oxidative
inhibits 1,5-lipoxygenase-dependent
lipid peroxidation. Free Radic Biol Med
2003; 35: 1203–1209.
hexadecanoate, a potent hyaluronidase
inhibitor. X-ray structure and molecu-
lar modeling of enzyme-inhibitor
complexes. J Biol Chem 2004; 279:
45990–45997.
stress. Am J Pathol 2004; 164: 1655– 15. Lapenna D et al. Inhibitory activity
1662.
of salicylic acid on lipoxygenase –
7. Rubin P, Mollison KW. Pharmaco-
therapy of diseases mediated by
dependent lipid peroxidation. Biochim 24. Cort WM. Antioxidant activity of
Biophys Acta 2009; 1790: 25–30.
tocopherols, ascorbyl palmitate, and
ascorbic acid and their mode of
action. J Am Oil Chem Soc 1974; 51:
321–325.
5-lipoxygenase pathway eicosanoids. 16. Raghavenra H et al. Eugenol – the
Prostaglandins Other Lipid Mediat
2007; 83: 188–197.
active principle from cloves inhi-
bits 5-lipoxygenase activity and
8. Cornicelli JA, Trivedi BK. 15-
Lipoxygenase and its inhibition: a
novel therapeutic target for vascular
leukotriene-C4 in human PMNL cells. 25. Pizarro F et al. Ascorbyl palmitate
Prostaglandins Leukot Essent Fatty
enhances iron bioavailability in iron-
fortified bread. Am J Clin Nutr 2006;
84: 830–834.
Acids 2006; 74: 23–27.
disease. Curr Pharm Des 1999; 5: 17. Bendich A. Handbook of free Radicals
11–20.
and Antioxidants in Biomedicine, Vol. 26. Han JH et al. Coating of peanuts with
9. Radmark O, Samuelsson B. 5-
lipoxygenase: regulation and possible
II. Boca raton, FL: CRC Press Inc.,
1989: 153–160.
edible whey protein film containing
alpha-tocopherol and ascorbyl palmi-
tate. J Food Sci 2008; 73: E349–E355.
involvement in atherosclerosis. Prosta- 18. Naidu KA. Vitamin
C in human
glandins Other Lipid Mediat 2007; 83:
health and disease is still a mystery? 27. Karmee SK. Biocatalytic synthesis of
162–174.
An overview. Nutr J 2003; 2: 7.
ascorbyl esters and their biotech-
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778
777

Ascorbic acid 6-palmitate and lipid peroxidation
Riyaz Mohamed et al.
nological applications. Appl Microbiol
Biotechnol 2009; 81: 1013–1022.
4-nitrocatechol. Acta Crystallogr
D
41. Liu ZQ et al. Making vitamin C
Biol Crystallogr 2004; 60(Pt 3): 613–
lipophilic enhances its protective effect
against free radical induced peroxi-
dation of low density lipoprotein. Chem
Phys Lipids 1998; 95: 49–57.
28. Bøyum A. Isolation of lymphocytes,
615.
granulocytes and macrophages. Scand 35. Aggarwal BB, Gehlot P. Inflammation
J Immunol Suppl 1976; 5: 9–15.
29. Aharony D, Stein RL. Kinetic mecha-
nism of guinea pig neutrophil 5-
and cancer: how friendly is the rela-
tionship for cancer patients? Curr 42. Weber
Opin Pharmacol 2009; 9: 351–369.
V
et al. New 3- and 4-
hydroxyfuranones as anti-oxidants
and anti-inflammatory agents. Bioorg
Med Chem 2005; 13: 4552–4564.
lipoxygenase. J Biol Chem 1986; 261: 36. Williams RN et al. Ascorbic acid
11512–11519.
inhibits the activity of polymor-
30. Naidu KA. Eugenol – an inhibitor of
lipoxygenase-dependent lipid peroxi-
dation. Prostaglandins Leukot Essent
Fatty Acids 1995; 53: 381–383.
phonuclear leukocytes in inflamed 43. Mohamed R et al. Neutralization of
ocular tissues. Exp Eye Res 1984; 39:
261–265.
local toxicity induced by Vipera
russelli phospholipase A2 by lipophilic
derivative of ascorbic acid. Curr Top
Med Chem 2011; 11: 2531–2539.
44. Prigge ST et al. Structure and mecha-
nism of lipoxygenases. Biochimie 1997;
79: 629–636.
37. Williams RN, Paterson CA. Modula-
tion of corneal lipoxygenase by ascor-
bic acid. Exp Eye Res 1986; 43: 7–13.
38. Pugh DM et al. Inhibitory effect of
L-ascorbic acid on the yield of prosta-
31. Lineweaver H, Burk D. Determination
of enzyme dissociation constants. J
Am Chem Soc 1964; 56: 11512–11519.
32. Thomsen R, Christensen MH.
MolDock:
a
new technique for
glandin F from the guinea-pig uterine 45. Clapp CH et al. Irreversible inactiva-
high-accuracy molecular docking. J
Med Chem 2006; 49: 3315–3321.
homogenates. Br J Pharmacol 1975;
tion of soybean lipoxygenase-1 by
hydrophobic thiols. Biochemistry 2002;
41: 11504–11511.
53: 469.
33. Schuettelkopf AW, van Aalten DMF. 39. Cameron E et al. Ascorbic acid and
PRODRG – a tool for high-throughput
crystallography of protein-ligand com-
cancer: a review. Cancer Res 1979; 39: 46. McMillan RM, Walker ER. Designing
663–681.
therapeutically effective 5-lipoxygenase
inhibitors. Trends Pharmacol Sci 1992;
13: 323–330.
plexes. Acta Crystallogr 2004; D 60(Pt 40. Cotelle P et al. Synthesis and antioxi-
8): 1355–1363.
dant properties of a new lipophilic
ascorbic acid analogue. Bioorg Med
Chem 2003; 11: 1087–1093.
34. Skrzypczak-Jankun E et al. Soybean
lipoxygenase-3 in complex with
778
© 2013 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, 66, pp. 769–778