Redox Report

Communications in Free Radical Research

ISSN: 1351-0002 (Print) 1743-2928 (Online) Journal homepage: http://www.tandfonline.com/loi/yrer20

The activity of propolis in the scavenging of

vitamin B2-photogenerated ROS

Mariela González, María L. Tereschuk, Susana Criado, Eugenia Reynoso,

Cecilia Challier, María Belén Agüero, Lorena Luna, Gabriela Ferrrari, María P.
Montaña & Norman A. García

To cite this article: Mariela González, María L. Tereschuk, Susana Criado, Eugenia Reynoso,
Cecilia Challier, María Belén Agüero, Lorena Luna, Gabriela Ferrrari, María P. Montaña
& Norman A. García (2015) The activity of propolis in the scavenging of vitamin B2-
photogenerated ROS, Redox Report, 20:6, 246-253

To link to this article: http://dx.doi.org/10.1179/1351000215Y.0000000033

Published online: 05 Feb 2016.

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 The activity of propolis in the scavenging of
vitamin B2-photogenerated ROS
Mariela González 1, María L. Tereschuk 1, Susana Criado 2, Eugenia Reynoso2,
Cecilia Challier 2, María Belén Agüero 3, Lorena Luna 3, Gabriela Ferrrari 4,
María P. Montaña 4Norman A. García 2
1
Departamento de Ingeniería de Procesos y Gestión Industrial, Facultad de Ciencias Exactas y Tecnología,
Univ. Nac. de Tucumán, Av. Independencia 1800, Argentina, 2Departamento de Química, Univ. Nac. de Río
Cuarto, Campus Universitario, Argentina, 3Instituto de Biotecnología. Inst. de Cs. Básicas, Universidad
Nacional de San Juan, Av. Libertador General San Martín 1109 (O), Argentina, 4Área de Química-Física, Univ.
Nac., de San Luis, Lavalle 1151, Argentina

Objectives: The study was focused on the activity of propolis from Amaicha del Valle, Argentina (ProAV) as a
Downloaded by [190.136.125.207] at 06:17 06 February 2016

promoter and scavenger of Riboflavin (Rf) – photogenerated reactive oxygen species (ROS).
Methods: Through a kinetic and mechanistic study, employing stationary and time-resolved photochemical
and electrochemical techniques, the protecting activity of ProAV was investigated.
Results: In the absence of light and Rf, ProAV exerted a relatively efficient inhibitory effect on 1,1-diphenyl-2-
picrylhydrazyl radicals and acts as a protector of artificially promoted linoleic acid oxidation. Under aerobic
visible-light-irradiation conditions, in the presence of Rf as the only light-absorber species, a complex picture
of competitive processes takes place, starting with the quenching of singlet and triplet electronically excited
states of Rf by ProAV. The species O2(1 g), O•− •
2 , H2O2, and OH are generated and interact with ProAV.
Discussion: ProAV behaves as an efficient ROS scavenger. It is scarcely photo-oxidized by interaction with
the mentioned ROS. Quantitative results indicate that ProAV is even more resistant to photo-oxidation than the
recognized antioxidant trolox. Two dihydroxychalcones, mostly present in the ProAV composition, are
responsible for the protecting activity of the propolis.
Keywords: Propolis, Vitamin B2 photogenerate, Antioxidant, Photodegradation

Introduction antioxidants has considerably increased the amount

A wide variety of potentially harmful agents such as
free radicals and reactive oxygen species (ROS) can
be generated in the human body and in different
kinds of foods. Organisms have several ways to
protect oneself against such species, and most of
these defensive measures involve the use of antioxi-
dants that inhibit or delay the oxidation of other mol-
ecules by preventing the initiation or propagation of
oxidizing chain reactions. There are two basic cat-
egories of antioxidants: synthetic and natural. Both
types are generally formed by compounds containing
phenolic structures.1–3 Synthetic antioxidants such as
butylated hydroxyanisole and butylated hydroxyto-
luene (BHT) have been employed since the early
1900s; however, there are restrictions on the use of
these compounds, due to their own potentially detri-
mental effects on human.4,5 Thus, interest in natural
Correspondence to: Mariela González, María L. Tereschuk, Departamento
de Ingeniería de Procesos y Gestión Industrial, Facultad de Ciencias
Exactas y Tecnología, Univ. Nac. de Tucumán, Av. Independencia 1800,
Argentina.
Email: magonzalez@herrera.unt.edu.ar; mtereschuk@herrera.unt.edu.ar
of research into different types of native substances.6,7
Useful natural antioxidants may turn out to be
found in the preparations of Apis bees. Apis bees
prepare a resin-like material named propolis to fill
holes and embalm predators that have died inside
their hives. This material is composed of plant resins,
bees wax, and secretions from worker bees’ head
glands. Importantly, propolis has shown biological
activity, and it has, therefore, been introduced it as a
potentially active principle for pharmaceutical
preparations.8–10
These biological properties are mainly ascribed to a
few substances, such as polyphenols, flavonoids, phe-
nolic acids, and their esters among which flavonoids
in particular are considered top contributors.11,12
Most research regarding antioxidants has in fact
been devoted to the antioxidant activity of flavonoids,
due to their ability to reduce free radical formation
and scavenge oxidative agents.13
Regarding to the study of antioxidants, it is possible
to use photochemical techniques.

© W. S. Maney & Son Ltd 2015

246 DOI 10.1179/1351000215Y.0000000033 Redox Report 2015 VOL. 20 NO. 6
 González et al.
Riboflavin (Rf ), also known as vitamin B2, is one of
the most important natural absorbers of visible light
and is present in a wide variety of biological systems
and foods. It has been postulated to be a sensitizer
for photopromoted reactions (mainly photo-oxi-
dations) that can produce physiological changes in sur-
rounding molecules.14–16 It is well known that Rf and
its derivatives generate the ROS singlet molecular
oxygen (O2(1Δg)) and superoxide radical anion (O•2 )
a
upon adequate photoirradiation.17–19 In the presence
of an electron donor, photogeneration of ROS hydro-
gen peroxide (H2O2) and hydroxy radical (OH•) by Rf
has also been reported.20,21
This manuscript describes the results of our sys-
tematic kinetic study on the potential reactive inter-
actions of Rf-photogenerated ROS with propolis and
two chalcones present in this molecule. Specifically,
we measured the activities of propolis both as a pro-
moter and scavenger of Rf-photogenerated ROS and
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evaluated its stability in the presence of degradative
species.
Experimental
Materials
A propolis sample (ProAV) was collected from bee-
hives in Amaicha del Valle (Tucumán, Argentina)
and stored at –18 °C.
Rf, superoxide dismutase (SOD) from bovine eryth-
rocytes, catalase (CAT) from bovine liver; mannitol, L-
tryptophan, beta-carotene, Folin–Ciocalteu’s phenol
reagent, and mono deuterated MeOH (MeOD) were
purchased from Sigma Chemical. Co.; Rose Bengal
(RB), sodium azide (NaN3), and furfuryl alcohol
(FFA), were provided by Aldrich, Merck, and Riedel
de Haën, respectively. The radical 1,1-diphenyl-2-
picrylhydrazyl (DPPH) was purchased from Aldrich.
HPLC-quality methanol (MeOH) was acquired from
Sintorgan. MeOD was employed in the time-resolved
determinations of O2(1Δg) phosphorescence to increase
its lifetime. Water was triply distilled. An ethanolic
extract of propolis ProAV (EE) was obtained as
described below, and 2′ ,4′ -dihydroxychalcone
(diOHCh) and 2′ ,4′ -dihydroxy-3′ -methoxychalcone
(diOHMeCh) were isolated22 in the laboratory of Dr
Alejandro Tapia of the University of San Juan in
San Juan, Argentina.
Methods
Preparation of propolis EE and isolation of diOHCh
and diOHMeCh
EE was prepared according to the method described
by Szliszka et al.23 The extraction yield was 69%.
Isolation of the chalcones diOHCh and diOHMeCh
from Zuccagnia punctata Cav. collected in Amaicha
del Valle was performed according to the methods of
Agüero et al.22
Activity of propolis in the scavenging of vitamin B2-photogenerated ROS
Total phenolic compounds
Total polyphenol (TP) contents in EE were determined
by the Folin–Ciocalteau colorimetric method, accord-
ing to Kumazawa et al.24 An EE solution was mixed
with the Folin–Ciocalteau reagent, and 0.5 ml 10%
Na2CO3. EE was analyzed at a final concentration
of 20 μg/ml. The total polyphenol content was
expressed as grams of gallic acid (GA) equivalent/
100 g of sample.
Free radical scavenging activity on DPPH
The free radical scavenging effects of ProAV and cate-
chin (as a reference) were assessed by the fading of an
ethanolic solution of DPPH radical as previously
reported by Lima et al.25 EE was assayed at concen-
trations of 100 μg/ml. The fading percentage was cal-
culated as follows:
Fading percentage
⎡ ⎛ Absorbance of sample ⎞⎤
⎢ ⎜ − Absorbance of blank ⎟⎥
⎢ ⎟⎥ × 100
= ⎢1 − ⎜⎝ Absorbance of DPPH ⎠⎥
⎣ ⎦
(1)
Color loss indicated the free radical scavenging
efficiency.
Antioxidant activity on linoleic acid oxidation
Analysis of antioxidant activity on linoleic acid oxi-
dation was carried out using a modified version of
the method developed by Ahn et al.26 We dissolved
5 mg of βbeta-carotene in a solution of 10 ml of
chloroform and 134 μl of linoleic acid, and then
added 380 μl of Tween 20. Test samples were evaluated
at a final concentration of 90 μg/ml, and BHT was
used as the reference. The percent antioxidant activity
was expressed relative to the initial absorbance of the
sample during a 300-minute incubation using equation (2):
 
At
% AA = × 100 (2)
Ao
where % AA is the percent antioxidant activity; Ao is
the initial absorbance at 470 nm and t = 0; At is the
absorbance at 470 nm and measurement time t (inter-
vals of 60 minutes for 300 minutes).
Absorption and fluorescence experiments
Ground-state absorption spectra were recorded using a
Hewlett Packard 8453 diode array spectrophotometer.
For stationary Rf fluorescence experiments, an RF
5301-PC Shimadzu spectrofluorimeter was used. The
excitation and emission wavelengths were 446 and
515 nm, respectively.
A classical Stern–Volmer treatment of the data was
applied using equation (3), where I 0 and I are the
respective fluorescence intensities of Rf in the presence
Redox Report 2015 VOL. 20 NO. 6 247
 González et al. Activity of propolis in the scavenging of vitamin B2-photogenerated ROS

and absence of the compound that eventually acts as a
quencher, Q, and Ksv (Ksv = 1kq × 1τ0) is the
Stern–Volmer constant for the quenching of excited
singlet Rf (1Rf*), with 1kq (see Scheme 1) and 1τ0
being the rate constants for the dynamic quenching
of 1Rf* and lifetime of 1Rf*, respectively.
passed through a 1270-nm interference filter and two
Wratten filters. The output of the detector was
coupled to a 400-MHz digital oscilloscope (HP
54504A) and to a personal computer for signal proces-
sing. Air-equilibrated solutions were employed in all
cases.

I0 Stationary photolysis and oxygen uptake

= 1 + Ksv[Q] (3)
I experiments
Stationary aerobic photolysis of aqueous solutions
containing ProAV and either Rf or RB were carried
Time-resolved O2(1Δg) phosphorescence detection
out in a PTI unit, provided with a high-pass mono-
The total quenching rate constant (kt, see Scheme 1)
chromator and 150-W Xe lamp irradiating at a wave-
for O2(1Δg) deactivation by Q was determined from
length of 445 ± 10 nm or in a home-made photolyser
the near-infrared time-resolved phosphorescence, and
for five non-monochromatic irradiation (150 W
employing equation (4), in which τ and τ0 are the
quartz-halogen lamp). In the latter case, 430-nm wave-
O2(1Δg) lifetimes in the presence and in the absence
length cutoff filters were used.
of Q, respectively.
The Rf- or RB-sensitized photo-oxygenation rates
τ0 of ProAV, diOHCh, or diOHMeCh were determined
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= 1 + kt τ 0 [Q] (4)
τ by evaluation of the initial slopes of oxygen consump-
The 532-nm wavelength output from a Nd:Yag laser tion vs. irradiation time, employing a specific oxygen
(Spectron) was used as the excitation source. The electrode (Orion 97–08).
emitted (O2(1Δg)) phosphorescence at 1270 nm was Reaction (chemical) rate constants for the inter-
detected at right angles using an Edinburgh action Q-O2(1Δg) (kr, see Scheme 1) were obtained as
Instruments EI-P germanium detector, after having described by Tratniek & Hoigné,27 from the ratio of

Scheme 1 Possible reaction pathways in an Rf-photosensitized process.

248 Redox Report 2015 VOL. 20 NO. 6

 González et al.
Figure 1 (A) Percent DPPH radical scavenging activity by catechin (•) and ProAV (□) as a function of substrate concentration. (B)
Percent decrease of the absorbance at 470 nm as a function of incubation time of a solution of beta-carotene/linoleic acid in the
absence (•) and in the presence of BHT (▴) and ProAV (■).
the first order slopes of the vitamin B6 family (B6D)
and reference consumption, each at the same concen-
tration, yielding kr/krR. The reference was FFA, with
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a krR = 1.2 × 108 M–1s–1.
The anaerobic rates of Rf photodecomposition were
determined in the absence and presence of different
concentrations of ProAv by the decrease in the 446-
nm absorption band of the vitamin following 5-
minute irradiation with visible light (wavelength
cutoff of 430 nm) employing MeOH as a solvent in
Ar-bubbled solution.
Results
Overall antioxidative activity of ProAV
The extraction yield for EE was 69.4% with a TP value
of 22.3 expressed as grams of GA per 100 g EE.
Figure 2 Changes in the UV-Vis absorption spectrum of
MeOH-H2O (1:1; v/v) solutions containing Rf (A446 = 0.4) and
57 μg/ml ProAV vs. A446 = 0.4 upon irradiation with visible
light. Inset (A) oxygen consumption vs. photoirradiation time
for MeOH-H2O (1:1; v/v) solutions containing Rf (A446 = 0.40)
plus (a) 38 μg/ml ProAV and (b) 38 μg/ml Trolox. Inset (B)
changes in the UV-Vis absorption spectrum in a MeOH
solution of RB (A630 = 0.4) and 64 μg/ml ProAV vs. RB (A630 =
0.4) upon irradiation with visible light. In all cases, a cutoff at
430 nm was used.
Activity of propolis in the scavenging of vitamin B2-photogenerated ROS
Fig. 1A shows the radical scavenging activity of
DPPH treated with various concentrations of ProAV,
as well as DPPH treated with various concentrations
of the radical trapper catechin.
Fig. 1B shows the similar antioxidant activities of
ProAV and the recognized artificial antioxidant
BHT28 on linoleic acid oxidation.
These preliminary assays roughly demonstrate the
potential of ProAV as an overall defensive agent
against aggressive oxidative species, but they do not
provide detailed information about the fate of ProAV
under oxidative stress conditions or data on the
kinetic and mechanistic aspects governing such
processes.
The Rf (A446 = 0.4)-sensitized photoirradiation of
air-equilibrated solutions of ProAV (57 μg/ml) in
MeOH-H2O (1:1;v/v) produces different changes in
the absorption spectrum of the propolis with respect
to the propolis sample without Rf. The irradiation
wavelength was greater than 430 nm, and in a spectral
region where only Rf absorbs the incident light. Fig. 2
shows the difference absorption spectra of the mixture
(Rf plus ProAV vs. Rf ) at different irradiation times.
Parallel photoirradiation of a solution containing
38 μg/ml ProAV, but otherwise the same as that
described above, gave rise to oxygen consumption.
Oxygen uptake was not observed in the absence of
light or ProAV. To standardize the evaluation of the
oxygen uptake rate, the same experiment was per-
formed by changing the ProAV concentration and
employing an identical W/W concentration of the
well-known artificial antioxidant Trolox.29 The
results are shown in the inset of Fig. 2.
These experiments suggest that ProAV can intercept
Rf-photogenerated oxidative species. It is particularly
relevant in this regard that (a) ProAV was chemically
affected by irradiation with visible light in the presence
of Rf as a dye sensitizer, (b) oxygenated species partici-
pated in the process, and (c) electronically excited
states of Rf participated in the overall interaction.
Redox Report 2015 VOL. 20 NO. 6 249
 González et al. Activity of propolis in the scavenging of vitamin B2-photogenerated ROS
Individualization of the active excited states of the
sensitizer, as well as determination of optimal sensitiz-
ing conditions, oxygen requirements, and local con-
centrations necessary for the effective interaction of
the involved reaction partners may help to elucidate
and interpret the reaction mechanism. These issues
were previously been discussed on the basis of the
well-known reaction sequence shown in Scheme 1.30
Q represents an electron donor species, namely
ProAV in most of the experiments in this study. In
some cases, the propolis sample was replaced with
diOHCh or diOHMeCh to compare the behaviors of
these substrates. Note that these flavonoid substrates
were the most abundant antioxidant components
detected in the ProAV sample.
According to Scheme 1, the absorption of incident
light promotes Rf to electronically excited singlet
(1Rf*) and triplet (3Rf*) states (5). Both states can
be quenched by Q through reactions (6) and (8) –
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the latter by means of an electron transfer process
that produces the respective semireduced and semiox-
idized forms (i.e. Rf•− + Q•+). The species Rf•− is
rapidly protonated. Reactions (10) through (12) rep-
resent the known generation of the ROS O•− 2 , OH ,
•
and hydrogen peroxide (H2O2) upon interaction of
the neutral radical of the vitamin with reactants natu-
rally present in the medium. Direct generation of O•− 2
through quenching of 3Rf* by dissolved oxygen (step
(7)) has been reported, with a quantum yield of
0.009.17 Reaction (13) represents the eventual oxi-
dation of Q by the generated ROS. An energy-transfer
reaction from 3Rf* to ground-state triplet molecular
oxygen O2(3Σ− g ) dissolved in the medium can occur,
Figure 3 Stern–Volmer plot for the quenching of 1Rf* by
ProAV. I 0 and I represent the stationary fluorescence
intensities of Rf (A446 = 0.1) in the absence and presence of
ProAV. Inset (A) (a) fluorescence spectrum of Rf (A446 = 0.1) in
MeOH; (b) the same in the presence of 200 μg/ml ProAV. Inset
(B) relative values for the rate of Rf (A446 = 0.2) degradation
upon irradiation with 445 ± 10-nm wavelength light in argon-
saturated methanolic solution in the absence (1 in the
abscissa axis) and in the presence of 15, 36, and 60 μg/ml
ProAV (2–4 in the abscissa axis, respectively).
250 Redox Report 2015 VOL. 20 NO. 6
producing the excited-state oxygen species O2(1Δg)
with a reported quantum yield of 0.49 in MeOH (reac-
tion (14)).31,32 This species can decay either by a col-
lision with surrounding solvent molecules (reaction
(15)) or by interaction with Q and/or Rf through an
exclusive physical interaction (reaction (16)) or chemi-
cal photo-oxidation (reaction (17)). The overall rate
constant for O2(1Δg) quenching (kt) is defined as the
sum of the rate constants for processes (16) and (17).
Quenching of 1Rf* and 3Rf*by ProAV
Rf yielded an intense fluorescence emission band cen-
tered at 515 nm (Fig. 3), with a emission quantum
yield (ΦF) of 0.25. In the presence of ProAV, the fluor-
escence quenching of 1Rf* produced a decrease in the
stationary emission intensity, but the shape of the flu-
orescence spectrum did not change (Fig. 3, inset A).
The main panel of Fig. 3 shows the quenching plot,
from which a Stern–Volmer constant of 7 × 10−4 L/
(mg s−1) was obtained when employing Rf in MeOH
(A446 = 0.1). The reported value for the fluorescence
lifetime of Rf in MeOH is 2 ns.17
Anaerobic photodegradation of Rf under visible
light predominantly proceeds through the triplet
state, and the rate can be estimated with absorption
spectroscopy. Irradiation of Ar-saturated solutions of
Rf in MeOH in the absence and presence of different
ProAV concentrations but otherwise under identical
experimental conditions showed that this rate
decreased as the concentration of the propolis
increased from 0 to 60 μg/ml (Fig. 3, inset B). At
these ProAV concentrations, practically no fluor-
escence quenching of Rf occurred. A simple calcu-
lation employing the Stern–Volmer constant for
fluorescence quenching indicates that ∼7% inhibition
of the 1Rf* emission should be expected for a ProAV
concentration of 60 μg/ml. A decrease of ∼78% in
the degradation rate of Rf was observed for this con-
centration of ProAV (Fig. 3, inset B). On this basis,
the experimental data strongly support the idea that
a long-lived triplet state, one that is an intermediate
in the photolysis of Rf, can be efficiently quenched
by relatively low concentrations of ProAV. The
reported lifetime for 3Rf* in MeOH is 12 μs.
Quenching of O2(1Δg) by ProAV and diOHCh
The interaction of ProAV and diOHCh with O2(1Δg)
was explored by employing time-resolved O2(1Δg)
phosphorescence detection and RB as a dye sensitizer
that generate O2(1Δg) in an exclusive fashion with a
quantum yield of ∼0.7 in MeOH.33 The RB-sensitized
photoirradiation of ProAV (RB (A630 = 0.4)) in
MeOH produced evident changes in the absorption
spectra of the propolis sample (Fig. 2, inset B). In
addition, the decrease of the emission lifetime of
O2(1Δg) in the presence of 68 mg/l ProAV in MeOD
 González et al.
Figure 4 Stern–Volmer plots for the quenching of O2(1Δg)
phosphorescence by diOHCh in MeOD in the presence (a)
and in the absence (b) of 10 mM NaOH. Inset: temporal decay
of the phosphorescence intensity of O2(1Δg) in the absence (a)
and presence (b) of 68 μg/ml ProAV in MeOD.
Photosensitizer: RB (A532 = 0.18).
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(Fig. 4, inset) unambiguously demonstrates the
O2(1Δg) quenching by the propolis sample with the
oxidative species. Rate constant values kt = 3.4 × 107
M−1 s−1 and 2.8 × 107 M−1 s−1 were obtained for
diOHCh and diOHMeCh in MeOD. These values
are on the same order of magnitude as the kt values
reported for other flavonoids.33 The eventual kr
value was tested by comparison with the well-known
reference compound FFA.
Practically no oxygen uptake by 0.5 mM diOHCh
or diOHMeCh plus RB (A630 = 0.4) in MeOH was
observed. Hence, they were both determined to have
a kr value ˂10−6M−1 s−1. The kr/kt ratio, which indi-
cates the fraction of overall quenching of O2(1Δg) by
the substrate that effectively leads to a chemical trans-
formation, indicates that the flavonoids are exclusive
physical quenchers of the oxidative species.
Figure 5 Bar graph of the relative rate values of oxygen
uptake by MeOH-H2O (1:1, v/v) solutions of Rf (A446 = 0.4) and
38 μg/ml ProAV as a function of photoirradiation time (cutoff =
430 nm) in the absence (1) and presence of 10 μg/ml CAT (2);
10 μg/ml mannitol (3); 10 μg/ml SOD (4); and 5 mM NaN3 (5).
Bar (6) corresponds to the relative rate value of oxygen uptake
by Rf (A446 = 0.4) alone.
Activity of propolis in the scavenging of vitamin B2-photogenerated ROS
The rate constant for diOHCh was also determined
in the presence of 10 mM NaOH. Under this con-
dition, the hydroxyl groups of the flavonoid are
ionized, and a significant increase in the kt value
should be observed.34 In fact a kt value of 1.4 × 108
M−1 s−1 was determined for diOHCh in the methano-
lic alkaline medium. Experimental kinetic data for the
interaction of diOHCh with O2(1Δg) are shown in the
main panel of Fig. 4.
The interactions of ProAV and diOHCh with ROS
To evaluate the potential interactions of Rf-photogen-
erated ROS with ProAV, we carried out oxygen con-
sumption experiments in the presence of specific
ROS interceptors. The presence of 10 mM NaN3,
1 μg/ml SOD, 1 μg/ml CAT, and 10 mM mannitol
was each observed to decrease the rate of oxygen
uptake of MeOH-H2O (1:1, v/v) solutions containing
Rf (A446 = 0.4) plus 38 mg/l ProAV as compared to
that recorded in the additives’ absences. Similar exper-
iments with ROS interceptors have been performed to
assess the participation of O2(1Δg), O•– 2 , H2O2, and
OH• in oxidative events.35–37 The enzyme SOD dismu-
tates the O•–
2 species (reaction (20)), whereas CAT
decomposes H2O2 (reaction (21)), mannitol deacti-
vates the species OH• (reaction (22)), and NaN3 phys-
ically quenches O2(1Δg) (reaction (16)) (Scheme 1, with
NaN3 instead of Q).
2O•− + −
2 + 2H + SOD  O2 ( Σg ) + H2 O2
3
(20)
2H2 O2 + CAT  2H2 O + O2 (3 Σg − ) (21)
•
OH + mannitol  deactivation (22)
Photoprotective effect of diOHMeCh toward
tryptophan oxidation
To test the eventual protective effect of diOHMeCh on
the ROS-mediated oxidation of proteins, we assessed
Rf-sensitized photo-oxidation of the amino acid
tryptophan (Trp).29 The rates of oxygen consumption
by 0.5 mM Trp, 0.5 mM diOHMeCh, and a mixture
of both substrates in MeOH were determined. These
rates are considered a measure of the global photo-oxi-
dative progress and were determined by monitoring up
to 15% of the conversion of the starting material. In
relative terms, the obtained rate values were 1, 0.31,
and 0.37 respectively. In other words, the rate of Trp
photodegradation in the presence of diOHMeCh
decreased by ∼63% relative to that in the absence of
the polyphenol.
It is well known that Trp is photo-oxidizable upon
Rf-sensitization.38 The degradative process operates
through a combination of radical-mediated and
O2(1Δg)mediated mechanisms. Garcia and Silva38
reported on the generation of O•– •
2 , H2O2, OH , and
O2( Δg) in Rf-sensitized Trp photodegradation, and
1
Redox Report 2015 VOL. 20 NO. 6 251
 González et al. Activity of propolis in the scavenging of vitamin B2-photogenerated ROS
we determined a kr = 3.5 × 107M−1 s−1 for reaction
(17) with this amino acid instead of Q.39
Discussion and conclusions
Overall antioxidant activity of ProAV
DPPH radical scavenging action and antioxidant
activity toward linoleic acid clearly demonstrate the
overall ability of ProAV to defend against such aggres-
sive detrimental agents.27,28 The antioxidative action
of ProAV is accompanied by its concomitant degra-
dation. This degradation mainly involves an exclu-
sively O2(1Δg)-mediated pathway in the case of RB
sensitization and a group of radical-driven reactions
in addition to the above-mentioned O2(1Δg) degra-
dation in the case of Rf-sensitization. Nevertheless,
the rate at which ProAV faded was kinetically moder-
ate, and less than twice the rate of fading in exper-
iments with Trolox (Fig. 2), a recognized artificial
antioxidant.29
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The promotion and quenching of ROS by ProAV
ProAV reacts with O2(1Δg), O•– •
2 , H2O2, and OH . The
• anisole and butylatedhydroxy toluene. J Am Oil Chem Soc
H2O2 and OH species were not observed to spon-
taneously form during Rf photoirradiation. Hence,
the initial action of ProAV could be considered as pro-
moting the generation of these ROS. The use of this
possible pathway has been demonstrated for a series
of electron donors in the presence of photoirradiated
Rf, including several flavonoids.30
The radical reaction initially produces RfH• and
ProAV•+ (reaction (8)). The latter species, representing
one or more electron-donating components of propo-
lis, possibly decays to products and/or reacts with
ROS generated in the medium. 3Rf* could be
quenched either by O2(3Σ− g ) (reaction (14)) or ProAV
(reaction (8)). Whether one of these processes is kine-
tically dominant depends on the respective concen-
trations of the quenchers and on the values of the
rate constants of reactions (8) and (14). Different flavo-
noids acting as efficient electron donors toward 3Rf*, a
species that can simultaneously produce O2(1Δg).40
The observation that under identical experimental
conditions, the detection tests for O2(1Δg) on one
hand and O•– •
2 , H2O2, and OH on the other hand,
clearly indicates that under these conditions, pathways
(8) and (14) are kinetically competitive to some extent.
Photoprotection of Trp photo-oxidation by
diOHCh
The dominant physical scavenging of O2(1Δg) rep-
resents a positive result arising in the context of oxi-
dative interactions between biomolecules. In this
case, the very low values for the kr/kt quotients exhib-
ited by diOHCh and by diOHMeCh are examples of a
desirable quality. The final result is the elimination of
O2(1Δg) without appreciable scavenger loss.
252 Redox Report 2015 VOL. 20 NO. 6
This activity protects proteins, DNA, and other cell
matrix components.41 This photoprotective effect
exerted by diOHCh was evident based on the clear
decrease in the rate of oxygen uptake of the system
containing Trp and chalcone compared to that of the
amino acid alone upon Rf sensitization.
Disclaimer statements
Contributors None.
Funding None.
Conflicts of interest There are no conflicts of interest.
Ethics approval Ethical approval is unnecessary.
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