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Molecular Biology Research Communications 2016;5(2):87-95 MBRC
*Address for correspondence: Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz, Iran.
Tel: +98-4113393926
Fax: +98-4113393929
E. mail: barzegar@tabrizu.ac.ir
pISSN 2322-181X eISSN 2345-2005
Original Article Open Access
Antioxidant activity of polyphenolic myricetin in vitro cell-
free and cell-based systems
Abolfazl Barzegar
Research Institute for Fundamental Sciences (RIFS), University of Tabriz, Tabriz,
The School of Advanced Biomedical Sciences (SABS), Tabriz University of Medical
Sciences, Tabriz, Iran
ABSTRACT
Myricetin (Myc) is one of the most important flavonoids in diet due to its abundance
in foods with the highest antioxidant activity. The antioxidant activity of Myc was
studied in cell-free and cell-based systems to evaluate the ROS protection efficiency of
Myc. The studies were based on the assessment of reducing power of Myc according to
ferric ion reduction and intracellular ROS level measurement by assaying the cellular
fluorescence intensity using dichlorodihydrofluorescein (DCF) probe as an indicator for
ROS in cells. Moreover, the antitoxic capability of Myc was assessed using MTT
method. Data indicated that intracellular ROS are highly toxic and applying low
concentration of Myc not only inhibited cellular ROS production but also was
accompanying with the protection of cells against the highly toxic and the lethal effects
of peroxide compounds. Because of strong correlation between cellular ROS and their
cell toxic properties, the higher antioxidant potency of Myc in cell medium resulted in
effectively blocking intracellular ROS and protecting cell death. This property is
achieved by the help of high polar solubility and cell membrane permeability of Myc.
Keywords: Myricetin; ROS; Antioxidant; MTT; FRAP
INTRODUCTION
Flavonoids compounds are a diverse group of plant metabolites and are found in a
wide variety of human foods [1, 2]. The flavonoids, as a large and complex group, are
the best defined groups of polyphenols in the human diet. They are a various and
complex group of compounds that are absorbed in the gut and linked to human health
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[1]. They contain a three-ring structure with two aromatic centers and a central
oxygenated heterocycle [3, 4]. Flavonoids can suppress carcinogenesis in animal models
and there is considerable interest in the biological effects of these compounds at the
cellular level [5]. Flavonoids are categorized into subgroups based on their chemical
structure: flavanones, flavones, flavonols, flavan-3-ols, anthocyanins and isoflavones.
Over 10,000 polyphenol compounds have been identified until now. However, only
few of them have been investigated in detail [5]. Most common flavonoids include
resveratrol, epigallocatechin 3-gallate, tyrosol, hydroxytyrosol, kaempferol, quercetin
and myricetin (Myc) [6-8]. Myc is a natural flavonol that exists in tea, different
vegetables, onions, berries, grapes and medical plants, with a unique chemical structure
[7]. Myc consists of two aromatic rings linked together with a heterocyclic pyrone ring
as displayed in Figure 1. It induces pancreatic cancer cell death [9], inhibits DNA strand
breakage [10], attenuates the ultraviolet B induced COX-2 expression and skin tumor
formation in a mouse skin model [11]. Treatment of insulin-resistant rats with Myc
leads to the alteration in the phosphorylation of the insulin receptor, with subsequent
effects on glucose-transporter subtype 4 translocation [12]. Results of in vitro studies
suggested that high concentrations of Myc can cause change in LDL cholesterol level
via an increase in the uptake of LDL cholesterol by white blood cells. A study
correlated high Myc consumption with lowered rates of prostate and pancreatic cancers
[13, 14]. Consequently, the compound Myc has a significant pharmacological
importance as a medicinal agent. The action of Myc at the molecular level is mainly
based on antioxidant effects. The antioxidant and radical scavenging activities of Myc
have been widely studied by different researchers for many years [15-18]. Results of
previous studies have showed that each Myc molecule is capable of scavenging
different radicals and it was more effective than α-tocopherol as an antioxidant in
liposomes [15]. However, the intracellular antioxidant behavior of Myc has not studied
yet.
O
O
OH
OH
OH
OH
OH
HO2
3
5
7
8
4
1
6
6'
2'
3'
5'
4'
Figure 1: The chemical structure of myricetin (Myc)
In this study, the ability of Myc to reduce and scavenge intra-cellular ROS was
evaluated using human MCF-7 cells as an in vitro cell model. In addition, cell-free
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system including “reducing power” has been applied for evaluating the effect of polar
solvent in ferric ion reduction capability of Myc.
MATERIALS AND METHODS
Materials: Chemical compounds including Myc, trichloroacetic acid, and cumene
hydroperoxide (CHP) were purchased from Sigma Chemical Co. Potassium ferricyanide
and ferric chloride were acquired from Merck. 2',7'-dichlorodihydrofluorescein
diacetate (DCFH2-DA) was obtained from Molecular Probes (Eugene, OR). Fetal
bovine serum was from GIBCO (Grand Island, NY). RPMI 1640, antibiotics and sterile
plastic for cell culture were from Flow Laboratory (Irvine, UK). Water treated in a
MicroMed-TKA system (conductivity < 0.1 µS cm-1), was used to prepare the solutions.
Stock solution of Myc was prepared using dimethylsulfoxide (DMSO) as a solvent from
Merck. Human MCF-7 breast cancer cells were obtained from the pharmaceutical
nanotechnology research center, Tabriz University of Medical Sciences, Tabriz, Iran
Cell cultures: Human MCF-7 breast cancer cells were grown in RPMI 1640
supplemented with 10% (v/v) fetal bovine serum, 100 U mL−1 penicillin, and 100 mg
mL−1 streptomycin. These cells were incubated at 37 ˚C with 5% CO2 and usually sub-
cultured once every 3 days.
DCF method for detection of the intracellular ROS: The intracellular ROS level in
MCF-7 cells was measured using DCF method. The details of DCF method were clearly
explained in our previous publications [19-21]. Briefly, the cell samples were incubated
one hour in the presence of 5 µM DCFH2-DA, followed by two times washing with
PBS. The washing procedure has been applied by the help of centrifugation with 2000
rpm to remove the extracellular DCFH2-DA. The oxidation of DCFH2-DA by
intracellular ROS resulted in fluorescent DCF which stains the cells. Hence, the
intracellular ROS generation of cells can be investigated using DCF method as an
indicator to detect and quantify intracellular produced reactive oxygen species. The
trapped fluorescent DCF dye inside the cells was used to evaluate and detect
intracellular ROS by spectrofluorometer. The intracellular ROS generator compound,
CHP, was used in 300 µM concentration. The incubation time for CHP was 2 min
followed by the fluorescent intensity changes by adding different concentrations of Myc
(0.0, 0.05, 0.1 and 0.2 µM). Experiments were done in triplicate and the mean value was
recorded.
MTT assay: In order to assay the antitoxic capability of Myc, methylthiazole
tetrazolium (MTT) method was utilized [19]. Viable cell numbers were recorded by
measuring the absorbance at a certain wavelength of 500–600 nm. MCF-7 cells were
seeded into 6-well plates until they reached 95–100% confluency. Then, they were
incubated for 30 min with different concentrations of Myc. Afterward, cells were
induced with cumene hydroperoxide (CHP) as a powerful cytotoxic ROS-generating
compound [21, 22]. The cells were incubated with MTT at a final concentration of 1
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mg/mL at 37 ˚C for one hour. Cells were separated by trypsinization followed by
centrifugation at 1200 rpm for 5 min. Then, the pellet was re-suspended in 300 mL
phosphate-buffered saline (PBS) and sonicated on ice for 20 seconds with an Ultrasonic
W-225R, at setting 4, and centrifuged in a microfuge at 13000 rpm for 10 min. In the
final step, the supernatant was discarded and the water-insoluble formazan assay
product was dissolved in DMSO and measured at 560 nm.
Ferric ion reducing antioxidant power (FRAP assay): FRAP activity was measured
according to our previously published method [21, 22]. The process by which the ferric
ion reduces into the +2 oxidation ferrous ion state (Fe2+) is known as reducing power
assessment. The reducing power of Myc was determined by analyzing its electron donor
potency according to ferric ion reduction. Potassium ferricyanide (1%) was shortly
incubated with different concentrations of Myc for 30 min at 50 °C in phosphate
buffered saline (PBS), then FeCl3 (0.1%) and trichloroacetic acid (10%) were added and
mixed. After the addition of trichloroacetic acid (10%) and FeCl3 (0.1%) the absorbance
at 700 nm was recorded as reducing power of Myc in PBS solution. Samples with
greater reducing power showed higher absorbance at 700 nm.
RESULTS
Electron-transfer reaction of Myc has been focused on Fe+3 reduction to Fe+2 in
aqueous solution that can be a significant indicator of the antioxidant activity known as
ferric ion reducing antioxidant power (FRAP). The FRAP assessment provides clear
information about the electron transfer potency of an antioxidant which is a simple,
rapid, and relatively inexpensive assay [22]. The absorbance at 700 nm was recorded as
a function of Myc concentrations in phosphate buffered saline (PBS) to monitor the
reduction of ferric ion to ferrous ion. Figure 2 indicates a significant linear relationship
between Myc concentrations and reduced amounts of iron (III) ions in polar solution.
Figure 2: Ferric iron (Fe3+) reduction capacity of Myc. The absorbance at 700 nm was recorded as a
function of Myc concentrations in phosphate buffered saline (PBS) to monitor the reduction of ferric ion
to ferrous ion.
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The ability of Myc to scavenge intracellular ROS was investigated using MCF-7
cells based on the DCF method. CHP is one of the oxidizing agents which has been
used as an intracellular source of reactive oxygen intermediates [19]. Myc combats
intracellular ROS generation which leads to decrease in the amount of DCF
fluorescence, as shown in Figure 3. In the absence of Myc, CHP causes substantial
oxidation of DCFH2 to DCF, leading to an increased rate of fluorescence intensity
change. Addition of Myc suppresses the intracellular fluorescence intensity at 485 nm
during 60 minutes. Consequently, the efficient suppression of intracellular ROS
production by Myc indicates that this compound enters the cells and acts with strong
radical scavenging potency in the polar intracellular environment. Moreover, the cell
membrane permeability of Myc was evaluated based on simultaneous DCF fluorescence
changes that are indicated in Table 1. More importantly, we observed an immediate
decrease in DCF fluorescence after the addition of 0.1 and 0.2 μM Myc which implies
the rapid penetration of Myc into cells and suppression of ROS generation. As a result,
Myc is able to diffuse through the cell membrane into the cells, where it prevents the
production of different ROS compounds.
Figure 3: Intracellular ROS determination by DCF method. The changes of fluorescence spectra were
monitored for the intracellular ROS during 60 min. The excitation wavelength, excitation slit and
emission slit were 485, 5 and 10 nm light path respectively. Samples were negative control (without
having antioxidant and ROS stimulator), positive control (having intracellular ROS inducer agent of
cumene hydroperoxide,CHP) and myricetin (having intracellular ROS inducer agent of CHP plus 0.5µM
myricetin).
Table 1: The membrane permeable efficiency of Myc to reduce intracellular ROS
Samples(µM) ΔF1 ΔF2 ΔF3 ΔF4
0 81 88 150 121
0.05 70 80 120 100
0.1 46 54 80 68
0.2 38 45 70 60
Note: Cell samples were induced by 300 µM radical generator CHP at the same time following the addition of
different concentrations of Myc (0.0, 0.05, 0.1 and 0.2 µM). DCF fluorescence changes were evaluated for 10
minutes and reported as ΔF/10 min for four different independent assays (ΔF1, ΔF2, ΔF3, ΔF4). High ΔF values
denote high intracellular ROS. None of the Myc samples tested, rose to fluorescence on their own.
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MTT method was used to evaluate the antitoxic properties of Myc against cytotoxic
and the lethal effects of CHP. As saying the antitoxic property of Myc has been
performed based on the suppression of cytotoxic effects of CHP. The reduction of
yellow MTT to purple formazan takes place only when mitochondrial reductase
enzymes are active, and therefore the amount of conversion can be directly accounted
for the percentage of viable (living) cells [19]. Figure 4 shows that in the absence of
CHP, 95% of cells are viable that reduce MTT compound. While, CHP is a highly toxic
and lethal compound for MCF-7 cells that causes 75% of cells to die. The presence of
Myc in samples almost suppressed the lethal effects of CHP. Hence, the role of Myc in
blocking the toxic and lethal effects of CHP has a strong correlation with its
intracellular ROS scavenging potency, as it was mentioned in Figure 3.
Figure 4: Cell viability assay using MTT method. Cells were treated with 5 µL (1:100) CHP. Negative
control indicates no addition of CHP/antioxidant and positive control denoting only presence of CHP.
Treating cells by 2 µM and 5 µM Myc during 30 min led to the decrease followed by complete
suppression of the toxic effects of CHP.
DISCUSSION
Reactive oxygen species, including free radicals, are formed by exogenous
chemicals and endogenous metabolic processes in the human body. Different diseases
have been associated with excessive ROS (Fig. 5), which are produced mostly in the
mitochondria as byproducts of cell respiration during mitochondrial electron transport
and other metabolic reactions [23, 24]. The suppressing/inhibiting intra-cellular ROS
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Most antioxidants are known radical chain breaking reactions based on their potency
in cell-free systems. Antioxidants preferentially localize to cellular compartments based
on solubility. Unfortunately, most of them are not effective within the cells, mainly
because of limited solubility, low permeability, and self-toxic lethal effects. The
different properties of Myc such as cell protection efficiency against toxic CHP,
intracellular ROS inactivator and ferric ion reduction power in polar medium proposed
the efficient flavonoid for the potential clinical, biological, and biotechnological
applications.
Acknowledgments: The authors would like to thank the Research Institute for
Fundamental Sciences (RIFS)-University of Tabriz for the financial supports.
Conflict of Interest: No competing interests are declared by the author.
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