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1Laboratory for Food Science and Metabolism, Department of Biochemistry, Faculty of Science University of Yaounde 1, Cameroon
2Laboratory of Biochemistry, Physiology and Pharmacology, Faculty of Medicine and Biomedical Science / UHC-University of Yaounde 1, Cameroon
Submission: May 01, 2019; Published: June 27, 2019
*Corresponding author: Kotue TC, Laboratory for Food Science and Metabolism, Department of Biochemistry, Faculty of Science University of Yaounde 1, Cameroon
How to cite this article: Kotue TC, Djote WNB, Marlyne M, Pieme AC, Kansci G, Fokou E. Antisickling and Antioxidant Properties of Omega-3 Fatty Acids EPA/DHA. Nutri Food Sci Int J. 2019. 9(1): 555752. DOI:10.19080/NFSIJ.2019.09.555752.
Background: Therapeutic treatment of Sickle cell disease (SCD) is complex and very expensive. However, natural products have been used to manage sickle cell crises. Omega-3 fatty acids EPA/DHA reduce the number of crisis in SCD.
Objectives: This study aimed to assess the antisickling and antioxidant properties of omega-3 fatty acids EPA/DHA.
Methods: The evaluation of the rates of inhibition of induced sickling by sodium metabisulfite 2% and the potential reversal of sickle cells into normal spherical erythrocytes was performed using microscopic enumeration of red blood corpuscles of the sickling. The evaluation of membrane stability effect, FRAP, DPPH°, and OH° assays was determined using colorimetric method.
Results: Sodium metabisulfite increased the sickling of RBCs from 26.3±1.6 to 79.42±5.2% during 3 hours. Omega-3 fatty acids EPA/DHA 0.2% showed the best antisickling (79.05±1.2%) and reversibility (64.82± 2.7%) rate; the best membrane stability compared to other concentrations. Omega-3 fatty acids EPA/DHA revealed an appreciable reducing power at 26.08±0.3mg FeII/100g. It also showed an inhibitory activity on free radicals of DPPH and hydroxyl radical at IC50 3.7±0.0 and 11.28±0.3mg/mL respectively.
Conclusion: Omega-3 fatty acids EPA/DHA have antisickling, anti-haemolytic and antioxidant properties. The results obtained are in addition to those of the authors who showed that Omega-3 fatty acids EPA/DHA reduce the number of crisis in SCD.
Omega 3 fatty acids are fats commonly found in marine and plant oils. They are polyunsaturated fatty acids (PUFA) with a double bond (C=C) starting after the third carbon atom from the end of the carbon chain. The fatty acids have two ends: - the acid (COOH) end and the methyl (CH3) end. The location of the first double bond is counted from the methyl end, which is also known as the omega (ω) end or the n end . Omega-3 long-chain polyunsaturated fat acid, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are dietary fats with an array of health benefits . They are incorporated in many parts of the body including cell membranes  and play a role in anti-inflammatory processes and in the viscosity of cell membranes [1,4]. EPA and DHA are essential for proper fetal development and healthy aging . EPA and DHA are also the precursors of several metabolites that are potent lipid mediators, considered by many investigators to be beneficial in the prevention or treatment of several diseases . Omega-3 fatty acids have been found to play a role in atherosclerosis
and peripheral arterial disease. It is thought that both EPA and DHA improve plaque stability, decrease endothelial activation, and improve vascular permeability, thereby decreasing the chance of experiencing a cardiovascular event . DHA is present in large amounts in neuron membrane phospholipids, where it is involved in proper function of the nervous system, which is why it is thought to play a role in Alzheimer’s disease .
The findings from a previous retrospective and cross-sectional study suggest that omega-3 fatty acids might be beneficial in Sickle Cell Disease (SCD) [9,10]. In fact, SCD is a group of autosomal recessive genetic blood disorders characterized by a single point mutation in the sixth codon of the β-globin gene. Under low oxygen tension, the resultant abnormal hemoglobin S polymerizes and causes rigid and sickle-shaped red blood cells. Sickle cell pain is the commonest manifestations of the disease in which episodic micro vessel occlusion in one or more sites induces tissue damage accompanied by severe pain and inflammation [11,12]. The
pain may be acute or chronic, somatic or visceral, unilateral or
bilateral, localized or diffuse . Painful crisis affects nearly
all patients often beginning in late infancy and recurring
throughout life  and it is the major cause of hospital
admissions . Moreover, adults who experience painful
crises more than three times per year tend to have shorter
life expectancies . Clinically, omega-3 fatty acid treatment
reduced the median rate of clinical vasoocclusive events, blood
transfusion. Subsequent to treatment there was a remarkable
reduction in the frequency of pain episodes requiring
hospital presentation . Similarly,  have demonstrated
a significant decrease in the number of crisis and steadystate
hemolysis in 16 Nigerians sickle cell patients treated
with Cod liver oil containing EPA and DHA. Several studies
have also demonstrated that dietary supplementation with
omega-3 fatty acids fish results in increased incorporation of
these fatty acids into the RBC membrane, which can influence
RBC deformability [19,20]. However, the antisickling and
antioxidant properties of omega-3 fatty acids EPA/DHA have
not been reported. Thus, this in vitro study was undertaken to
investigate the effect of omega-3 fatty acids EPA/DHA on blood
obtained from patients with sickle cell disease.
Omega-3 fatty acids EPA/DHA pure fish oil capsules
were bought in a pharmacy. Each capsule contains 750μL in
proportion EPA/DHA (3v/2v). The following concentrations of
0.2% v/v, 0.4v/v and 0.6 v/v of the oil were obtained by diluting
EPA/DHA in normal saline diluted ethanol (1:4 in normal
Sickle cell blood (HbS) samples from 8 males and 8 females,
aged 16 and above were obtained from Central Hospital
of Yaounde-Cameroon. The permission of Regional Bioethics
Committee of Centre with authorization CEN° 00504/
CRERSHC/2018 was obtained for all the research procedures.
A total of Two milliliters (2ml) of venous blood samples were
collected from each patient in the sodium EDTA tubes and
stored for the experiment.
About 100μL of HbS blood cell suspensions were mixed
with 100μL of 2% sodium metabisulfite solution (Na2S2O5)
and incubated at 37 °C. The time course of the sickling of HbS
erythrocytes was analyzed microscopically according to the
method described by . The number of cells was counted
every one hour after take 10μL of the mixture diluted 200 times
using Marcano liquid. The number of cell was counted every
one hour and the percentage of sickling cells was calculated
using the formula:
A curve of percentage of sickling in function of time was
realized. This permitted the deduction of maximum time
necessary to obtain maximum sickling.
For the assay, in 100μL of sickled cell blood (HbS) preincubated
with 2% Na2S2O5 w as a dded t o 1 00μL o f s olution
of different concentrations of omega-3 fatty acids EPA/DHA
(0.2% v/v; 0.4v/v and 0.6 v/v) previously prepared.
Each mixture was incubated at 37 °C for 3 h (time necessary
to obtain maximum sickling). After incubation, 10μL of the
mixture was diluted 200 times using Marcano liquid. 10μL
of each sample was examined under the light microscope and
both sickle cells and total cells were counted from five different
fields of view across the slide. For the negative control, the
solution containing the extract was replaced by the saline
solution. The percentage of sickling inhibition was calculated
using the formula:
is the % of sickling of the mixture SS blood and 2 % Na2S2O5.
is the % of sickling of the mixture SS blood 2% Na2S2O5 and omega-3 fatty acids EPA/DHA with each concentration.
For the reversibility assay, in 100μL of sickle cell blood (HbS)
was added to 100μL of solution of different concentrations of
omega-3 fatty acids EPA/DHA.
The experiment was followed as mentioned above. The
percentage of sickling cells was calculated after every 1 hour
till the maximum reversibility of sickling was attained.
The percentage of sickling was determined the same
way. Calculation was done after every 1 hour until maximum
reversibility of sickling was attained. These percentages
permitted to calculate the rate of reversibility of sickling
according to the following formula.
R is the reversibility rate (%)
R0 is the initial percentage of sickling and Rn is the
maximum percentage of sickling obtained with different
concentrations of omega-3 fatty acids EPA/DHA.
The evaluation of the erythrocyte membrane stability
effect of omega-3 fatty acids EPA/DHA was done using a method
proposed by . The osmotic f ragility of t he erythrocytes is
based on the measurement of the stabilizing effect of their
membrane after 24 h of incubation using omega-3 fatty acids
EPA/DHA at different concentrations. Varying concentrations
of normal saline were prepared (0 - 0.85% NaCl). To 5.05mL
reaction vessel containing 4.5mL of each NaCl concentration
and 0.5ml of each EPA/DHA concentration, 0.05ml SS blood
was added. The mixture was incubated at 37 °C for 24 h and
then centrifuged at 3000 rpm for 15min. The optical density
of the supernatant was read at 540nm against blank made
of 0.85% buffered saline concentration. The percentage of
hemolysis was calculated using the formula below:
Results were presented graphically as percent hemolysis
plotted against the concentration of NaCl.
Evaluation of the total antioxidant activity by ferric
reducing antioxidant power assay (FRAP) was done according
to . For this evaluation, 0.1mL of omega-3 fatty acids EPA/
DHA (2mg/mL) was mixed with 3mL of freshly prepared FRAP
reagent. After incubation (up to 5minutes) in darkness at room
temperature (~25 °C), absorbance was read against a suitable
blank at 593nm. The test was carried out in triplets. The
concentration of omega-3 fatty acids EPA/DHA was calculated
using the standard equation obtained by using standard
FeSO4. The final results were expressed in mg of Fe (II)/100g of
omega-3 fatty acids EPA/DHA. Gallic acid was used as control.
The scavenging effect of omega-3 fatty acids EPA/DHA on
DPPH radical was estimated by method described by . A
solution of 0.1mM DPPH in methanol was prepared, and 1.0mL
of this solution was mixed with 3.0mL of omega-3 fatty acids
EPA/DHA solution of varying concentrations. The reaction
mixture was vortexed thoroughly and left in the dark at room
temperature for 30 min. The absorbance of the mixture was
measured spectrophotometrically at 517nm. Gallic acid was
used as standard. The ability to scavenge DPPH radical was
calculated by the following equation:
The inhibition percentages calculated permitted the
realization of curves, percentage inhibition in function of
extract concentration. The test was carried out in triplets.
A curve of % DPPH bleaching activity versus concentration
was plotted using OriginPro 8 Software to determine IC50
concentration that account for 50% inhibition.
Hydroxyl radical assayed as described by . To 1.5mL
of each dilution of the extra omega-3 fatty acids EPA/DHA
solution of varying concentrations, we added successively
60μdL of FeCl3 1Mm; 90μL of 1.1 o-Phenanthroline 1Mm;
2.4mL of phosphate 0.2M, pH 7.8 and 150μL of H2 02 0.17M.
The mixture is then homogenized and incubated at normal
temperatures for 5minutes. After 5 minutes absorbance was
read at 560nm against the blank. Gallic acid was used as the
standard. The antiradical activity of omega-3 fatty acids EPA/
DHA expressed in percentage of inhibition of the hydroxyl
radical was determined following the formula:
The inhibition percentages calculated permitted the
realization of curves percentage inhibition in function of
omega-3 fatty acids EPA/DHA concentrations using OriginPro
8 Software. These curves were used to determine IC50
concentration that account for 50% inhibition. The test was
carried out in triplets.
The results were expressed as mean ± standard deviation.
Data was analyzed using Analysis of Variance (ANOVA)
of Kruskall-Wallis with the software Sigma Start version
3.01A analysis software. Statistical data were considered
significantly different at 95% confidence interval (p <0.05).
Sodium metabisulfite 2%, added to sickled red blood cells
induced sickling of red blood cells (Figure 1).
At the beginning, sickling percentage was 26.3±1.6 and
after 3 hours of incubation sickling increased to 79.42±5.2%
and remained constant with time. The maximum number
of sickled cells was obtained after 3 hours, suggesting that
this is the time necessary to obtain maximum sickling. In
this study, the percentage of sickling RBCs obtained after
3 hours of incubation is lower than 96.5% and 80% values
obtained respectively by  at the same period and  after
1 hour. MBS 2% create hypoxic conditions for RBCs leadind
to loss of the morphology and sickled erythrocytes. In vitro
deoxygenating of RBCs by MBS (2%) caused progressive
aggregation and polymerization of the individual hemoglobin
molecules [27,28]. The process of gelation (polymerization) of
hemoglobin molecules increases the formation of sickling cells.
After calculating the rate of inhibition of sickling, it was
realized omega-3 fatty acids EPA/DHA (0.2%) significantly
(p<0.05) inhibited sickling giving inhibition rates of
79.05±1.2% more than 70.541±2.6% and 69.24±2.24% for
omega-3 fatty acids EPA/DHA concentrations of 0.4 and .06%
respectively. According to reversibility rate, we noticed a
significant (p<0.05) reversal effects with omega-3 fatty acids
EPA/DHA (0.2%) giving rate of 64.82±2.7 more than 59.77±2.5
and 59.69±2.3 for omega-3 fatty acids EPA/DHA concentrations
of 0.4 and 0.6% respectively (Table 1).
Indeed, these results show that omega-3 fatty acids EPA/
DHA have antisickling properties at a certain percentage.
Furthermore, it has been reported that Tiger nut oil (Cyperus
esculentus) and black seed oil (Nigella sativa) containing
omege-3 in their profile also possess antisickling properties
. Their effect may also be due to the presence of omega-3.
However, none mode of action of this effect has been
Figure 2 represents the effect of omega-3 fatty acids EPA/
DHA on membrane stability. Hemolysis decreased for all
concentrations in the presence of sodium chloride (0.9%) until
13.54% for omega-3 fatty acids EPA/DHA (0.2%) significantly
(p<0.05) upper than that exhibited by omega-3 fatty acids
EPA/DHA 0.4 and 0.6% and the control.
The effect of omega-3 fatty acids EPA/DHA on the membrane
stability of RBCs can be evaluated by comparing the haemolysis
rates of untreated and treated sickle RBCs with omega-3 fatty
acids EPA/DHA at different concentrations. A decrease in the
percentage of haemolysis as a function of omega-3 at different
concentrations was generally noted. This decrease is related to
the appreciable protective effect of omega-3 fatty acids EPA/
DHA on the erythrocyte membrane, hence, their resistance against haemolysis. Furthermore, membrane stability of
RBCs was dose-independent with increasing concentration
of omega-3. The membrane of erythrocytes is predominantly
made up of lipids and the proportions of the different types
of lipids affect membrane integrity, structure and function
. For example, the erythrocytes of rabbits fed on diet rich
in omega-3 fatty acids EPA/DHA show greater resistance to
lysis in hypotonic saline relative to red blood cells of control
animals on normal diet; suggesting that omega-3 fatty acids
confer protection against haemolysis .
Table 1 show the evaluation of the antioxidant properties
of omega-3 fatty acids EPA/DHA using gallic acid as references.
Omega-3 fatty acids EPA/DHA exhibited antioxidant potentials
and an average reducing power at 26.08±0.3mg FeII/100g of
Omega-3 fatty acids EPA/DHA after carrying out FRAP but
significantly (p<0.05) lower compared to gallic acid (mg
FeII/100g). It also showed a significant (p<0.05) inhibitory
activity on free radicals of 2,2-Diphenyl-1picrylhydrazyl
(DPPHo) and hydroxyl radical (HOo) at IC50 3.7±0.0mg/mL and
11.28±0.3mg/mL respectively but remains lower than gallic
acid. (Table 2)
It was also found that the Tiger nut oil (Cyperus esculentus)
and black seed oil (Nigella sativa) treatments resulted in an
increase in the antioxidant presence of sickle cell samples
when tested in vitro . I n f act, oxidative phenomena play a
significant role in the physiopathology of sickle cell disease.
Sickle RBCs produce greater quantities of superoxide radical,
hydrogen peroxide (H2O2) and hydroxyl radical than do normal
RBCs . Despite the evident beneficial effects of n-3 fatty
supplementation for patients with SCD, there was a lingering
concern that the fatty acids, because of their high double bond
index and susceptibility to peroxidation , might exacerbate
the inherent oxidative stress associated with the disease. We
discover with this study that omega-3 fatty acids EPA/DHA
has a global antioxidant capacity and is active on 2,2-Diphenyl-
1picrylhydrazyl and hydroxyl radicals.
Omega-3 fatty acids EPA/DHA have antisickling, antihemolytic
and antioxidant properties. The results obtained are
in addition to those of the authors who showed that Omega-3
fatty acids EPA/DHA was effective at reducing the frequency
and severity of haemolysis, vaso-occlusive episodes, severe
anemia, and blood transfusion. The mode of action of these
properties studied will be the next point of focus within this