Protective Effect of Myricetin on Proteins and Lipids of Erythrocytes Membranes
Aida A. Mahmoud
Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone), a naturally occurring flavonol, is a potent scavenger of Reactive Oxygen Species (ROS) and effectively prevent erythrocyte oxidation. The protective effect of myricetin on proteins and lipids of erythrocytes membranes was investigated. Erythrocytes membranes were subjected to oxidative stress by incubating them with 10-5 M tert-butylhydroperoxide; this caused a significant increase in membrane protein carbonyl group content and membrane lipid peroxides while caused a significant decrease of membrane total thiol group and Na+, K+-ATPase activity. The presence of myricetin in micromolar concentration in the incubation medium decreased significantly protein carbonylation, lipid peroxidation and caused an increase in total thiol group and Na+, K+ ATP-ase activity.
Received: October 03, 2012;
Accepted: March 11, 2013;
Published: April 13, 2013
Reactive Oxygen Species (ROS) are oxidants usually produced during the course
of metabolism by most cells of the body. The intracellular antioxidant systems,
either, enzymatic or nonenzymatic react with these oxidant converting them to
nonreactive species (Srinivasan and Avadhani, 2012).
Oxidative stress occurs as a result of depletion or inadequacy of antioxidant
systems causing damage to intracellular biomolecules including proteins, lipids
and nucleic acids (Ayer et al., 2010). Oxidative
stress may take place under normal conditions but its incidence increases with
age and during disease conditions as the efficiency of antioxidant and repair
mechanisms decreases (Gil et al., 2006).
As erythrocytes carry oxygen to various organs, they are exposed to ROS. The
presence of oxygen, polyunsaturated fatty acids and iron induce lipid peroxidation
and protein oxidation causing loss of functions of membrane biomolecules like
membrane-bound enzymes and receptors and alter the fluidity of the membranes
(Matough et al., 2012).
Erythrocytes possess defense mechanisms against toxic species, but the efficiency
of these mechanisms decreases in conditions that cause overproduction of ROS.
Therefore, the additional supplementation of antioxidants is important for the
protection against oxidative stress and other disease conditions, such as atherosclerosis,
ischemia, inflammation, cancers, cardiovascular and neurological diseases (Rice-Evans,
Flavonoids are polyphenolic compounds found in considerable levels in dietary
plants. The flavonoid family is divided into a number of sub-groups; namely
flavonols, flavones, flavan-3-ols, isoflavones, flavanones and anthocyanidins
(Crozier et al., 2000). The antioxidant activities
of polyphenols of dietary origin are well documented and may be responsible
for various health profits (Brownson et al., 2002).
The penetration of polyphenolic compounds into cell membranes prevents oxidation
and its sequel.
|| Chemical structure of myricetin
The achievement of an efficient protection against membrane oxidation by means
of phenolic compounds may be due to their ability to incorporate into biological
membranes (Suwalsky et al., 2009).
Myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4 chromenone), is a
flavonol present in some dietary plants and in walnuts. Myricetin may be occurring
as a glycoside (Fig. 1) (Miean and Mohamed,
In this study, the protective effect of myricetin on proteins and lipids of
erythrocytes membranes subjected to oxidative stress (by incubating with 10-5
M tert-butylhydroperoxide) was examined, through measuring the membrane protein
carbonyl group content, lipid peroxides, total thiol group and the activity
of Na+, K+- ATPase in presence and absence of myricetin.
MATERIALS AND METHODS
This study was carried out in Biochemistry Department, Sohag Faculty of Medicine in accordance with the guidelines of the ethical committee of Sohag University, during the year 2011.
Chemicals: Myricetin, Guanidine hydrochloride, 5, 5'-Dithio-Bis (2 Nitrobenzoic acid) and tert-butylhydroperoxide were purchased from Sigma (St. Louis, MO, USA). Other chemicals and reagents were purchased from MERK (Darmstadt, Germany).
Blood samples: Venous blood samples (5 mL) were obtained by venipuncture
in heparin from 15 healthy volunteers after taking their informed written consent.
The blood was centrifuged at 1,800 g for 10 min at 4°C. After the removal
of plasma, buffy coat and upper 15% of the packed Red Blood Cells (RBCs), the
RBCs were washed twice with cold PBS (0.9% NaCl, 10 mM Na2HPO4,
pH 7.4) (Dodge et al., 1963).
Erythrocyte ghost preparation: Erythrocyte ghosts from leukocyte-free
RBCs were prepared according to the method of Dodge et
al. (1963). RBCs pellets were hemolyzed on ice with 9 volumes of 5 mM
phosphate buffer (pH 7.4) for an hour and centrifuged for 20 min at 15000 g
and 4°C. The process was continued until the washing buffer became colorless.
The white ghosts finally resolved in equal volumes of PBS. Protein concentrations
were determined according to the method of Lowry et al.
Determination of membrane protein carbonyl: Erythrocyte membrane protein
carbonyls were measured according to the procedure of Levine
et al. (1990), as follows, two tubes of 0.25 mL membrane suspension
were taken, one as a test and the other as a control. 1.0 mL of 10 mM 2, 4 dinitrophenyl-hydrazine
(DNPH) prepared in 1.25 M HCl was added to the test sample and 1.0 mL of 2.5
M HCl alone was added to the control sample. The contents were mixed and incubated
in the dark at room temperature for 1 h and shaken intermittently every 15 min.
Then 1. 25 mL of 20% TCA (w/v) was added to both tubes and the mixture left
in ice for 10 min. The tubes were then centrifuged at 3,500 rpm for 20 min to
obtain the protein pellet. The supernatant was carefully aspirated and discarded.
This was followed by a second wash with 10% TCA. Finally, the precipitates were
washed three times with 1 mL of ethanol: ethyl acetate (1:1, v/v) to remove
unreacted DNPH and lipid remnants. The final protein pellet was dissolved in
1 mL of 6 M guanidine hydrochloride (GuHCL) and incubated at 37°C for 10
min. The insoluble materials were removed by centrifugation. Carbonyl concentrations
were determined from the difference in absorbance at 370 nm between the test
and the control samples, with ε370 = 22000 M-1 cm-1.
Carbonyl levels were expressed as nM mg-1 protein.
Determination of membrane lipid peroxides: Lipid peroxides were measured
using the method described by Draper and Hadley (1990)
based on thiobarbituric acid reactivity (TBARs), as following; 200 μL of
membrane suspension was mixed with a solution containing 15% trichloroacetic
acid (w/v), 0.38% thiobarbituric acid and 0.25 N HCL and heated at 100°C
for 10 min in boiling water bath. After centrifugation, the absorbance was taken
at 535 nm and the results were expressed as nmol TBARs mg-1 protein.
Determination of thiol group: It is determined according to thiol/disulfide
reaction of thiol and Ellman's reagent (5,5'-dithiobisnitrobenzoic acid) (Hu,
1994). Fifty microliter of membrane suspension was mixed with 1 mL 0.1 M
Tris, 10 mM EDTA pH 8.2, constituting the blank reaction and assessed at 412
nm. After that, we added 40 μL 10 mM DTNB in methanol and the absorption
read at 412 nm after stable colour formation (1-3 min). The concentrations of
thiol groups were calculated using a molar coefficient of 13.600 M1cm-1
. Thiols were expressed as nM mg-1 protein.
Na+, K+-ATP-ase activity assay: The activity of
Na+, K+-ATPase was assayed according to the method of
Bartosz et al. (1994), as follows, 200 μL
of membrane suspension was incubated for 1 h at 37°C in 1 mL of a reaction
mixture consisting of 3 mM ATP, 5 mM MgCl2, 140 mM NaCl, 14 mM KCl,
1 mM EDTA and 10 mM tris-HCl. The reaction was stopped by addition of 1 mL of
15% trichloroacetic acid and shaken vigorously, then centrifuged at 3000 g for
15 min. The released Pi in the supernatant was measured by the method of Fiske
and Subbarow (1925). Total ATPase activity was expressed as μM of Pi
released per hour per mg protein. This assay was repeated in the presence of
200 μM methyldigoxin, an inhibitor of Na+K+-ATPase
activity. The activity of Na+K+-ATPase was subsequently
determined by subtracting total ATPase activity in the presence of methyldigoxin
from enzyme activity in the absence of the inhibitor drug.
Induction of oxidative stress: Erythrocyte ghosts were incubated with t-BHP, 100 μM mL-1 alone or with t-BHP and myricetin, 50 μM mL-1 (myricetin is added first followed by t-BHP after 10 min) for 1 h and protein carbonyls, lipid peroxides, total thiol group and Na+, K+ ATP-ase activity were measured as described previously.
Statistical analysis: Results were expressed as Mean±SD. Statistical analysis of the data were performed using GraphPad Prism (Graph Pad Software, San Diego, CA, USA).
Obtained results revealed that incubation of erythrocytes membranes with 10-5
M t-BHP caused an increase in the level of lipid peroxide and protein carbonyl,
meaning that the antioxidant systems present in the membrane cannot overcome
the oxidative stress induced by t-BHP. Decrease in Na+, K+
ATP-ase activity meant that the oxidative stress caused malfunction of membrane
enzymes. Myricetin, a naturally occuring flavonol possessed a powerful antioxidant
capacity. Myricetin in concentration of (50 μM mL-1) protected
the membrane proteins and lipids and restored the Na+, K+
ATP-ase activity. The incubation of erythrocytes membranes with t-BHP caused
a significant increase of membrane protein carbonyl from 1.5±0.4-6.2±0.7
0.4 nM mg-1 protein (the increase was about 4.1 folds), the presence
of myricetin in concentration of (50 μM mL-1) caused a significant
decrease in membrane protein carbonyl reached to 2.7±0.4 nM mg-1
protein (the decrease was about 2 folds) (Fig. 2). Lipid peroxides
increased from 0.43±0.01-5.8±0.68 5 nM mg-1 protein,
(the increase was about 13.5 folds) in absence of myricetin, the presence of
myricetin decreased significantly lipid peroxides to 2.1±0.42 nM mg-1
protein (the decrease was about 2.8 folds) (Fig. 3).
||Effect of myricetin on erythrocyte membrane carbonyl group
content of oxidatively stressed RBCs
|| Effect of myricetin on erythrocyte membrane lipid peroxides
of oxidatively stressed RBCs
Total thiol group concentrations decreased significantly from 28.7±5.4-13.4±3.2
(nM mg-1 protein) in absence of myricetin (the decrease was about
2.14 folds) and reached 20.4±4.1 nM mg-1 protein in presence
of myricetin (the increase was about 1.4 folds) (Fig. 4).
Na+, K+ ATP-ase activity decreased from 0.59±0.07-0.17±0.05
(μM Pi mg-1 protein h-1) in absence of myricetin
(the decrease was about 3.4 folds) and decreased to 0.39±0.06 μM
Pi mg-1 protein h-1 in presence of myricetin (the increase
was about 1.5 folds) (Fig. 5, 6).
|| Effect of myricetin on erythrocyte membrane thiol group of
oxidatively stressed RBCs
|| Effect of myricetin on erythrocyte membrane total ATPase
of oxidatively stressed RBCs
|| Effect of myricetin on erythrocyte membrane Na-K ATPase of
oxidatively stressed RBCs
|| Effect of incubation with t-BHP alone and t-BHP and myricetin
on the variables (***,**,*p<0.05)
|*, ** Significant at p<0.05 and <0.001
The most affected parameter by oxidative stress was lipid peroxides which
increased by about 13.5 folds followed by protein carbonyl; the increase was
about 4.1 folds, then Na+, K+ ATP-ase activity; the decrease
was about 3.4 folds and finally total thiol group the decrease was about 2.14
folds (Table 1).
This investigation revealed an antioxidant protective effect of myricetin (3,5,7-Trihydroxy-2-(3, 4, 5-trihydroxyphenyl)-4-chromenone) on proteins and lipids of erythrocytes membranes subjected to oxidative stress, as it decreased the formation of protein carbonyl, lipid peroxides and caused an increase in total thiol group and Na+, K+ - ATPase activity.
Plant flavonoids including myricetin are powerful therapeutic agents, effective
against free radical mediated diseases. Flavonoids can bind to tryptophan residues
in erythrocytes membranes ghosts causing antioxidant and antihemolytic effects
(Chaudhuri et al., 2007). In accordance with
the results of this study, several reports showed the protective effect of flavonoids
on erythrocyte membrane lipids, proteins and thiols (Coskun
et al., 2005; Rizvi and Mishra, 2009; Pandey
and Rizvi, 2010).
Flavonoids intake was inversely related to the mortality from congestive heart
disease and the incidence of myocardial infarction. The protection offered by
flavonoids was found to be due to their antioxidant activity. The aromatic rings
of the flavonoid molecule allow the donation and acceptance of electrons from
free radical species (Suwalsky et al., 2009).
In addition to reacting with free radicals, flavonoids are able to regenerate
the traditional antioxidant vitamins, C and E (Vinson et
Myricetin may act as a hydrogen donor and can suppress free radical processes
at three stages, the formation of superoxide ion, the generation of hydroxyl
radicals in the fenton reaction and the formation of lipid radicals (Moridani
et al., 2003; Rice-Evans et al., 1996).
It may also suppress lipid peroxidation by regenerating other antioxidants,
such as α-tocopherol, through reduction of α-tocopheroxyl radicals
(Rice-Evans et al., 1996). The activity of myricetin
against free radicals is due to the presence of O-dihydroxy (catechol) structure
of the B ring, the 2,3-double bond in conjugation with a 4-oxo-function in ring
C and both of 7- and 5-additional hydroxyl groups in ring A (Fig.
1) (Khanduja and Bhardwaj, 2003) Erythrocytes and
erythrocytes membranes are more susceptible to oxidation due to their continuous
exposure to high oxygen tension. Erythrocyte membranes are an excellent model
for membrane studies because of the simplicity, availability and ease of isolation
of them (Kolanjiappan et al., 2002).
Protein oxidation occurs upon attack of ROS forming carbonyl groups. The amino
acids; lysine, arginine, proline and histidine are the most liable to modification.
(Lemarechal et al., 2006). The accumulation of
protein carbonyls w as found to be associated with a number of diseases, such
as lateral sclerosis, Alzheimers disease, respiratory distress syndrome,
muscular dystrophy and rheumatoid arthritis (Dalle-Donne
et al., 2003) Lipid peroxidation have been shown to cause profound
alterations in the structural organization and functions of the cell membrane-bound
enzymes and loss of essential fatty acids (Lam et al.,
Thiols act as antioxidants reacting with ROS, so protect cells against the
damage induced by them. The intracellular and extracellular redox states of
thiols play a an important role in keeping protein structure and function, regulation
of enzymatic activity of transcription factors and antioxidant protection (Wloodek,
Na+, K+-ATPase (EC 220.127.116.11), is a member of P-type family
of active cation transport proteins. It transports excessive Na+ ions
out from the cells as it transports three Na+ ions out of the cell
and two K+ ions into the cell using the energy derived from hydrolysis
of one molecule of ATP (Vlkovicova et al., 2008)..
The stable ion content is needed for normal physiological activity. ATPases
are among the enzymes that can be attacked by ROS leading to decreased enzyme
activity (Rodrigo et al., 2007). In accordance
with the protective effect of myricetin on Na+, K+-ATPase
found in this study, (Lam et al., 2007), showed
a protective effect of polyphenolic compounds present in dietary plants on Na+,
K+-ATPase of erythrocytes membranes (Lam et
al., 2007). From the previous study, we concluded that myricetin (3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4-chromenone)
can protect proteins and lipids of erythrocyte membranes, so can be used as
an additional supplementation for protection against oxidative stress and other
pathologies, such as atherosclerosis, ischemia, inflammation, cancers, cardiovascular
and neurological diseases.
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