Redox Report

The activity of propolis in the scavenging of Vitamin B2-photogenerated ROS

--Manuscript Draft--

Manuscript Number: RER-11-302R1

Full Title: The activity of propolis in the scavenging of Vitamin B2-photogenerated ROS

Article Type: Research Article

Keywords: Propolis; Vitamin B2 photogenerate; Antioxidant; Photodegradation

Corresponding Author: Mariela Gonzalez, PhD

FACET-UNT
San Miguel de Tucumán, Tucumán ARGENTINA

Corresponding Author Secondary

Information:

Corresponding Author's Institution: FACET-UNT

Corresponding Author's Secondary

Institution:

First Author: Mariela Gonzalez, PhD

First Author Secondary Information:

Order of Authors: Mariela Gonzalez, PhD

María L Tereschuk, PhD

Susana Criado, PhD

Eugenia Reynoso, PhD

Cecilia Challier, PhD

María B Aguero, PhD

Lorena C Luna, PhD

Gabriela Ferrari, PhD

María P Montaña, PhD

Norman A García, PhD

Order of Authors Secondary Information:

Abstract: Objectives: The study was focused on the activity of propolis from Amaicha del Valle,
Argentina (ProAV) as a 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 DPPH 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(1g), O2.- , H2O2 and OH. are generated and interact with
ProAV.
Discussion: ProAV behaves as an efficient ROS scavenger. It is scarcely
photooxidized by interaction with the mentioned ROS. Quantitative results indicate that
ProAV is even more resistant to photooxidation than the recognized antioxidant trolox.
Two dihydroxychalcones, mostly present in the ProAV composition, are responsible for
the protecting activity of the propolis.

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Title page

The activity of propolis in the scavenging of Vitamin B2-

photogenerated ROS

Mariela González1*, María L. Tereschuk1*, Susana Criado3, Eugenia Reynoso3,

Cecilia Challier3, María Belén Agüero4, Lorena Luna4, Gabriela Ferrrari2,

María P. Montaña2 and Norman A. García3

1
Departamento de Ingeniería de Procesos y Gestión Industrial. Facultad de
Ciencias Exactas y Tecnología. Univ. Nac. de Tucumán, Av. Independencia 1800,
(4000) Tucumán. Argentina.
2
Área de Química-Física, Univ. Nac. de San Luis, Lavalle 1151, (5700) San Luis.
Argentina.
3
Departamento de Química, Univ. Nac. de Río Cuarto, Campus Universitario,
(5800) Río Cuarto. Argentina
4
Instituto de Biotecnología. Inst. de Cs. Básicas, Universidad Nacional de San
Juan, Av. Libertador General San Martín 1109 (O), 5400 San Juan. Argentina.

(*) Corresponding author. MG: magonzalez@herrera.unt.edu.ar; MLT:

mtereschuk@herrera.unt.edu.ar; Tel.: +54 381 4364093.

Response to reviewer comments

Departamento de Ingeniería de Procesos y Gestión Industrial

Universidad Nacional de Tucumán Dra. Mariela González
Tucumán – República Argentina e-mail: magonzalez@herrera.unt.edu.ar

San Miguel de Tucumán, April 4th, 2015

Professor Nicholas Hunt

Editor of Redox Report

Dear Professor Hunt,

This is a replying letter referred to the revised version of the Ms RER-11-302

entitled The activity of propolis in the scavenging of Vitamin B2-
photogenerated ROS, by M. González et al.
All comments and suggestions raised by you and by the Reviewers have been
taken into account, and the changes introduced are listed below.
An additional version of the Ms, with all the changes highlighted has been also
uploaded.
We acknowledge all the mentioned constructive criticisms that contributed to an
improved version of the Ms.
Editor:
The use of English was carefully revised with the assistance of a native English
speaker.

Reviewer #1
1. The authors should clearly establish what is new and relevant and a resume
of the quantitative results should be given in a Table.
We agree with Reviewer #1 in the sense that the results should be presented in a
more quantitative fashion, in particular those related to the antioxidative properties
of propolis towards photogenerated ROS. Nevertheless we could not arrive to a
satisfactory an elegant arrangement of these results in a table format. So instead
we did our best in make a more quantitative presentation of the results in the new
version of the Ms. Besides, an explicative paragraph on the novelty and relevance
of the work and a sort of resume of the quantitative results were included in a new
section named “Final remarks”. (Lines 415-423).

2. The authors should discuss (or at least mention) the variability between
different Pro samples in order to get some understanding of the results relevance.
A short discussion on the possible variability between different Pro samples was
included in lines 87-91.

3. The ms is too long and very difficult to read. Part of the problem is that the
results are obtained with two sensitizers (Rf and RB), three substrates (ProAv and
diOHCh and lately Trp), several ROS and different reference compounds (Trolox,
BHT, catequine). This precludes a meaningful quantitative comparison of the
reported results.
The reasons for the employement of the different sensitizers and the different
substrates are now clearly explained. Besides, and in our opinion, a meaningful
quantitative comparison is now given along the paper.

4. The results should be quantified avoiding statements such as “at least

partially”, “to a minor extent, almost similar”, “an almost similar fashion” given in the
abstract.
All the mentioned, and other similar statements, have been replaced by more
defined expressions, along the Ms. Please, see also response to comment No1.

5. How can be compared the reactivity of a pure compound with

that of a complex extract? What it is concluded from Fig. 1 ?
6. The lines for ProAV in Figure 1 A are completely arbitrary and can not be
employed for quantitative conclusions. More points with their errors are
needed.

This is an answer to comments 5. and 6. from Reviewer 1.

We totally agree with points 5. and 6. raised by Reviewer 1. The only purpose of
Fig. 1 is to qualitatively show some evidence of ProAV as a possible interceptor
of oxidative and/or radical species. This was made in comparison to the known
behavior of catechin and linoleic acid as classical radical trapper and oxidizable
target respectively. In none of the cases the objective was an accurate evaluation
of antioxidant power of the propolis. This statement was clearly established in the
present version of the Ms (lines 204-226). In other words, Figure 1 A and B
simply constitutes an introductory presentation of the potential antioxidant activity
of ProAV which was, along the paper, systematically studied in the presence Rf-
and RB-photogenerated ROS.
.

7. Line 146 , “difference absorption spectrum ?

The expression “difference absorption spectrum” was replaced by “absorption
spectrum”.
 8. Clarify the meaning of “individual absence of light and ProAV?
The meaning is absence of light or absence of ProAV. The phrase was
rewritten.

9. Scheme 1 is very confuse and must avoid unbalanced chemical equations

(see Eqn. 7) .
We agree with Reviewer 1. Scheme 1 was totally rewritten. Unbalanced
equations were corrected. We think that Scheme 1 is necessary in order to
illustrate the processes of ROS photogeneration and quenching.

Reviewer #3:

1. Review of the contemporary literature should be updated.

This point was addressed and references 8-12 were updated (lines 439 and
451) .

2. Few recent literature regarding riboflavin photosensitization (J. Phys. Chem.

A. 2014, 118, 3934; J. Photochem. Photobiol. B. 2013, 119, 60; J.
Photochem. Photobiol. B. 2012, 113, 22) should be discussed in the
manuscript. For the mechanistic pathways of other sensitizers: Phys. Chem.
Chem. Phys. 17 (2015) 166.
The mentioned papers were included and briefly discussed in the context of
mechanistic pathways of phtosensitization (lines 96-102 and 338-345).

3. The authors have mentioned Scheme 2 in line no. 118. However, the
scheme 2 is missing in the manuscript.
The inclusion of “Scheme 2” was a typing error.

4. The authors should specify the wavelength at which the antioxidant activity
of DPPH is measured.
It was specified.

5. In line no. 233, the rate constant of diOHCh increases in alkaline medium.
The mechanism should be discussed more clearly.
We did our best in clarifying the discussion on this point in the new version of
the Ms (line 318-323).

6. In line no. 287, the authors addressed that ProAV initially enhances
ROS generation. Details are necessary.
This point was more extensively discussed and clarified in lines 400-408.

We hope that the present version of the Ms will be suitable for publication in
Redox Report.
Sincerely yours,
Mariela González
Manuscript (.doc/.docx)
Click here to download Manuscript (.doc/.docx): The activity of propolis in the scavenging of Vitamin B2 manuscript.docx

1 1. INTRODUCTION

2 A wide variety of aggressive agents such as free radicals and reactive oxygen

3 species (ROS) can be generated in the human body and in different kind of foods.

4 Some defences play a protective role and most of these defences are antioxidants.

5 It means compounds that inhibit or delay the oxidation of other molecules by

6 preventing the initiation or propagation of oxidizing chain reactions. There are two

7 basic categories of antioxidants, namely synthetic and natural. In general, both

8 categories are formed by compounds containing phenolic structures.1-3 Synthetic

9 antioxidants such as butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene

10 (BHT) have been employed since the beginning of 1900. However, there are

11 restrictions on the use of these compounds, in connection to human health.4,5

12 Thus, the interest in natural antioxidants has increased considerably the research

13 activity on different types of native substrates.6,7

14 Apis bees prepare a product named propolis to fulfil holes and to embalm

15 predators died inside the hives, composed of plant resins, bee wax and secretions

16 of the head glands of workers bees. Propolis have shown biological activity which

17 has introduced it as a potential active principle for pharmaceutical preparations.8-10

18 These functional properties are mainly ascribed to a few substances, such as

19 polyphenols of flavonoids and phenolic acids and their esters among which

20 flavonoids in particular are considered as top contributors.11,12

21 Most research work has been devoted to the antioxidant activity of flavonoids,

22 which is due to their ability to reduce free radical formation and to scavenge

23 oxidative agents.13 Riboflavin (Rf), is one of the most important natural visible-light
24 absorbers present in a wide variety of live systems and foods. It has been

25 postulated as a sensitizer for photopromoted reactions –mainly photooxidations–

26 that can produce physiological changes in the surrounding molecules.14-16 It is well

27 known that Rf and derivatives generate the ROS singlet molecular oxygen

28 (O2(1g)) and superoxide radical anion (O2•─) with upon adequate

29 photoirradiation.17,18,19 In the presence of an electron donor also the

30 photogeneration of the ROS hydrogen peroxide (H2O2) and hydroxy radical (OH•)

31 has been reported. 20,21

32 In the present work we discuss results of a systematic kinetic study on the potential

33 reactive interactions of Rf-photogenerated ROS with propolis and with two

34 chalcones presents in it. By this way we evaluated both the activity of propolis as a

35 promoter and scavenger of Rf-photogenrated ROS and its own stability in the

36 presence of the degradative species.

37 2. EXPERIMENTAL

38 2.1 Materials

39 Propolis sample (ProAV) was collected from beehives situated in Amaicha del

40 Valle (Tucumán, Argentina). It was stored at – 18 ºC.

41 Riboflavin (Rf), superoxide dismutase (SOD) from boverythrocytes, catalase from

42 bovine liver, mannitol, L-tryptophan, β – carotene, Folin - Ciocalteu’s phenol

43 reagent, and Mono deuterated MeOH (MeOD) were purchased from Sigma Chem.

44 Co. Rose Bengal (RB), Sodium azide (NaN3) and Furfuryl alcohol (FFA),were

45 provided by Aldrich, Merck and Riedel de Haën respectively. The radical 1,1-

46 Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Aldrich. Methanol (MeOH)

47 HPLC quality, was from Sintorgan. MeOD was employed in the time-resolved

48 determinations of O2(1g) phosphorescence in order to enlarge the lifetime of this

49 species. Water was triply distilled. Ethanolic Extract of propolis ProAV (EE) was

50 obtained as described below and 2’,4’- dihydroxychalcone (diOHCh) and 2’,4’-

51 dihydroxy-3’-methoxychalcone (diOHMeCh) were isolated22 in the Laboratory of Dr.

52 Alejandro Tapia from the University of San Juan (UNSJ), San Juan, Argentina.

53 2.2 Methods

54 2.2.1 Preparation of Propolis EE and isolation of diOHCh and diOHMeCh

55 Preparation of EE was performed according to Szliszka et al.23 The extraction yield

56 was 69%. Isolation of Chalcones diOHCh and diOHMeCh from Zuccagnia punctata

57 Cav. collected in Amaicha del Valle, was performed according to methods of

58 Agüero et al.22

59 2.2.2 Total Phenolic compounds

60 Total polyphenol contents in EE were determined by the Folin-Ciocalteau

61 colorimetric method, according to Kumazawa et al.24 EE solution was mixed with

62 Folin-Ciocalteau reagent and 0.5 mL of 10% Na 2CO3. EE were evaluated at the

63 final concentration of 20 μg/mL. Total polyphenol content was expressed as grams

64 gallic acid equivalent/ 100 gram of sample.

65 2.2.3 Free Radical Scavenging Activity on DPPH

66 Free radical scavenging effects of ProAV and catechin as reference were

67 assessed by the fade of a ethanolic solution of 1,1-diphenyl-2-picrylhydrazyl radical

68 as previously reported by Lima et al.25 EE was assayed at concentrations of 100

69 μg/mL. Fade percentage was calculated as follows (Eq. (3)):

sor ance o sample - sor ance o lan

70 a e percentage [ - ( )] (3)
sor ance o

71 The loss of color indicated the free radical scavenging efficiency.

72 2.2.4 Antioxidant activity on linoleic acid oxidation

73 This analysis was carried out using the method developed by Ahn et al.26 with

74 some mo i ications. We issolve 5 mg o β-carotene in 10 mL of chloroform, 134

75 µL of linoleic acid and 380 µL of Tween 20 was added. Test samples were

76 evaluate at the inal concentration o 9 μg/mL and BHT were used as the

77 reference. The antioxidant activity % was expressed relative to the initial

78 absorbance of the sample during 300 min incubation using eq. (4):

79 ( t / o) (4)

80 AA % = antioxidant activity percent

81 Ao = Initial Absorbance at 470 nm and t = 0

82 At = Absorbance at measurement time at 470 nm (intervals of 60 min during 300

83 min).

84 2.2.5 Absorption and fluorescence experiments

85 Ground state absorption spectra were registered employing a Hewlett Packard

86 8453 diode array spectrophotometer. For the stationary Rf fluorescence

87 experiments, a RF 5301-PC Shimadzu spectrofluorimeter was used. Excitation

88 and emission wavelengths were 446 and 515 nm, respectively.

 89 A classical Stern-Volmer treatment of the data was applied through Eq. (1), where

90 I0 and I are the respective fluorescence intensities of Rf in the presence and in the

91 absence of the eventual quencher Q, and Ksv (Ksv= 1kq x 10) is the Stern-Volmer

92 constant for the quenching of excited singlet Rf (1Rf*), being 1kq (see Scheme 1)

93 and 10 the respective rate constants for the dynamic quenching of 1Rf* and the

94 lifetime of 1Rf*.

95 I0 / I= 1 + Ksv [Q] (1)

96 2.2.6 Time resolved O2(1g) phosphorescence detection (TRPD)

97 The total quenching rate constant (kt, see Scheme 1) for O2(1g) deactivation by an

98 eventual quencher Q, was determined by near-IR time resolved phosphorescence,

99 employing eq. (2), being  and 0 the respective O2(1g) lifetimes in the presence

100 and in the absence of Q.

101 0 /  = 1+ kt 0 [Q] (2)

102 The 532 nm output from a Nd:Yag laser (Spectron) was used as the excitation

103 source. The emitted (O2(1g)) phosphorescence at 1270 nm was detected at right

104 angles using a Edinburgh EI-P Germanium detector, after having passed through

105 1270 nm-interference and two wratten filters. The output of the detector was

106 coupled to a 400 MHz digital oscilloscope (HP 54504A) and to a personal

107 computer for signal processing. Air equilibrated solutions were employed in all

108 cases.
109 2.2.7 Stationary photolysis and oxygen uptake experiments

110 Stationary aerobic photolysis of aqueous solutions containing ProAV plus either Rf

111 or RB were carried out in a PTI unit, provided with a high pass monochromator and

112 150 W Xe lamp, irradiating with 44510 nm, or in a home-made photolyser for 5

113 non-monochromatic irradiation (150 W quartz-halogen lamp). In this case cut-off

114 filters (430 nm).

115 The Rf- or RB-sensitized photooxygenation rates of ProAV, diOHCh or diOHMeCh

116 were determined by evaluation of the initial slopes of oxygen consumption vs.

117 irradiation time, employing a specific oxygen electrode (Orion 97–08).

118 Reactive (chemical) rate constants for the interaction Q-O2(1Δg) (kr, see Scheme 2)

119 were obtained as described by Tratniek and Hoigné27, from the ratio of the first

120 order slopes of B6D and reference consumption, each at the same concentration,

121 yielding kr/krR. The reference was FFA, with a krR =1.2 x 108 M–1s–1.

122 The anaerobic rates of Rf photodecomposition were determined in the absence

123 and in the presence of different ProAv concentrations by the decrease in the 446

124 nm absorption band of the vitamin upon 5 min irradiation with visible light (cut off

125 430) employing MeOH as a solvent, in Ar-bubbled solutions.

126 3. RESULTS

127 3.1 ProAV overall antioxidative activity.

128 The extraction Yield for EE was 69.4 % with total phenolic value (TP) of 22.3

129 expressed as grams of gallic acid (GA) per 100 g EE.

130 The comparative runs between the radical scavenger action on DPPH by ProAV

131 and the well-known radical trapper catechin were performed. Results are shown in

132 Figure 1A.

133 Please, insert Figures 1A and 1B here.

134 Following, in Figure 1B is shown the antioxidant activity on linoleic acid oxidation.

135 The ability of ProAV is nearly the same as that of the recognized artificial

136 antioxidant BHT.28

137 These preliminary assays roughly demonstrate the potentiality of ProAV as an

138 overall defensive agent against aggressive oxidative species. Nevertheless, none

139 of them bring detailed information about the fate of ProAV under conditions of

140 oxidative stress and even less about kinetic and mechanistic aspects governing

141 such as processes.

142 The Rf (A446 = 0.4)–sensitized photoirradiation of air-equilibrated solutions of ProAV

143 (57μg/ml) in MeOH-H2O (1:1;V/V) produces different changes in the absorption

144 spectrum of the propolis. The irradiation wavelength was higher than 430 nm, a

145 spectral region where only Rf absorbs the incident light. Figure 2 shows the

146 difference absorption spectrum of the mixture (Rf plus ProAVvs. Rf) at different

147 irradiation times.

148 Please insert Fig. 2 here

149 Parallel photoirradiation of a similar solution to the above described one (with 38

150 μg/ml ProAV in this case) gave rise to oxygen consumption. No oxygen uptake was

151 observed in the individual absence of light and ProAV. In order to standardize the
152 evaluation of the rate of oxygen uptake, the same described experiment was

153 performed by changing ProAV by and identical W/W concentration of the well-known

154 artificial antioxidant Trolox.29 Results are shown in Figure 2, inset.

155 From these pieces of experimental evidence, ProAV appears as a potential

156 interceptor of Rf-photogenerated oxidative species and three observations are

157 particularly relevant in the context: a) ProAV is chemically affected by the visible

158 light-irradiation in the presence of Rf as a dye-sensitizer; b) oxygenated species

159 participate in the process and c) electronically excited states of Rf seems to

160 participate in the overall interaction.

161 Individualization of the active excited states of the sensitizer, optimal sensitizing

162 conditions, oxygen requirement and local concentration necessary for the effective

163 interaction of the involved reaction partners may help to elucidate and interpret the

164 reaction mechanism. It was discussed on the basis of the following well known

165 reaction sequence (Scheme 1).30 Q represents an electron donor species, namely

166 ProAV in most of the experiments of the present contribution. In some cases the

167 propolis was replaced by diOHCh or diOHMeCh, in order to compare the behaviour

168 of these substrates, being the flavonoids the most abundant a priori antioxidant

169 components detected in the ProAV sample.

170

171 Please, insert Scheme 1 here

172 According to Scheme 1, the absorption of incident light promotes Rf to

173 electronically excited singlet (1Rf*) and triplet (3Rf*) states, (5). Both states can be

174 quenched by Q through reactions (6) and (8). The latter by means of an electron
175 transfer process by which the respective semireduced and semioxidized forms, Rf•-

176 + Q•+, are produced. The species Rf•- is rapidly protonated. Reactions (10)-(12)

177 represent the known generation of the ROS O2•-, OH• and hydrogen peroxide

178 (H2O2) and upon interaction of the neutral radical of the vitamin with adequate

179 reactants naturally present in the medium. Direct generation of O2•- through the

180 quenching of 3Rf* by dissolved oxygen (step (7)) has been reported, with quantum

181 yield of 0.009.17 Reaction (13) represents the eventual oxidation of Q by the

182 generated ROS. An energy transfer reaction from 3Rf* to ground state-triplet

183 molecular oxygen O2(3-g) dissolved in the medium can occur, producing the

184 excited state oxygen species O2(1g) with a reported quantum yield of 0.49 in

185 MeOH (reaction (14).31,32 This species can decay either by collision with

186 surrounding solvent molecules (reaction (15)) or by interaction with Q and/or Rf

187 through an exclusive physical (reaction (16)) or chemical (photooxidation, reaction

188 (17)). An overall rate constant for O2(1g) quenching (kt) is defined as the sum of the

189 rate constants for processes (16) and (17).

190 3.2 Quenching of 1Rf*and 3Rf*by ProAV

191 Rf presents an intense fluorescence emission band, centered at 515 nm. The

192 reported emission quantum yield (ΦF) is 0.25. In the presence of ProAV, the

193 fluorescence quenching of 1Rf* produces a decrease in the stationary emission

194 intensity, but the shape of the fluorescence spectrum does not change, as shown

195 in Fig 3, inset A. The main Figure shows the quenching plot from which a Stern-

196 Volmer constant of 7 x 10-4 L/mg. s-1 was obtained in MeOH, employing Rf (A446 =
197 0.1) . The reported value for the fluorescence lifetime of the vitamin in MeOH is 2

198 ns.17

199 Please, insert Figure 3 here

200 It is known that anaerobic photodegradation of Rf under visible light irradiation

201 predominantly proceeds through the triplet state, and the rate of the process can

202 be estimated by absorption spectroscopy. Comparative irradiations of Ar-saturated

203 solutions of Rf in MeOH under identical experimental conditions, in the absence

204 and in the presence of different ProAV concentrations showed that this rate

205 decreased in the presence of the propolis in the range 0-60 g/mL, employing

206 MeOH as a solvent (Figure 3, inset B). At the said ProAV concentrations,

207 practically no fluorescence quenching of Rf occurs. A simple calculation employing

208 the Stern-Volmer constant for the fluorescence quenching, indicates that an

209 inhibition of ca. 7% in the emission of 1Rf* should be expected for a ProAV

210 concentration of 60 g/mL. In parallel, a decrease of ca. 78% in the degradation

211 rate of Rf was observed for this concentration of ProAV (Figure 3, inset B). On this

212 basis, the experimental data strongly supports the idea that a long-lived triplet

213 state, intermediate in the photolysis of Rf, can be efficiently quenched by relatively

214 low ProAV concentrations. The reported lifetime for 3Rf* in MeOH is 12 s.

215 3.3 Quenching of O2(1g) by ProAV and diOHCh

216 The eventual interaction of ProAV and diOHCh with O2(1g) was explored

217 employing TRPD and RB as a dye sensitizer which generates O2(1g) in an

218 exclusive fashion with a quantum yield of ca. 0.7 in MeOH.33 The RB- sensitized

219 photoirradiation of ProAV (RB (A630 = 0.4)) in MeOH produced evident changes in

220 the absorption spectra of the propolis shown in Figure 2, inset B. Besides, the

221 decrease of the emissive lifetime of O2(1g) in the presence of 68 mg/L ProAV in

222 MeOD (Figure 4, inset) unambiguously demonstrate the quenching of O2(1g) by

223 the propolis with the oxidative species. Rate constant values kt = 3.4 x 107 M-1s-1

224 and 2.8 x 107 M-1s-1 were obtained for diOHCh and diOHMeCh in MeOD. It is in the

225 same order of magnitude of reported kt values for other flavonoids.33 The eventual

226 kr value was tested by comparison with the well-known reference compound FFA.

227 Practically no oxygen uptake by the systems 0.5 mM diOHCh or diOHMeCh plus

228 RB (A630 = 0.4) in MeOH was observed. Hence a kr value ˂ 10-6M-1s-1 can be

229 ascribed for both of them. The kr/kt ratio, which indicates the fraction of overall

230 quenching of O2(1Δg) by the substrate that effectively leads to a chemical

231 transformation indicates that the flavonoids are practically exclusive physical

232 quenchers of the oxidative species.

233 The rate constant for diOHCh was also determined in the presence of 10 mM

234 NaOH. Under these conditions the hydroxyl groups of the flavonoid are ionized,

235 and a significant increase in the kt value should be observed.34 In fact a kt value of

236 1.4 x 108 M-1s-1 was determined for diOHCh in the methanolic alkaline medium.

237 Experimental kinetic data for the interaction diOHCh-O2(1Δg) is shown in Fig 4,

238 main.

239 Please insert Figure 4 here

240 3.4 The interaction ProAV and diOHCh with ROS

241 In order to evaluate the potential interaction of Rf photogenerated ROS with ProAV

242 oxygen consumption experiments in the presence of specific ROS interceptors

243 were carried out. The presence of 10 mM NaN3; 1 μg/ml SOD; 1 μg/ml CAT and 10

244 mM mannitol decrease the rate of oxygen uptake of MeOH-H2O (1:1, v/v) solutions

245 containing Rf (A446 = 0.4) plus 38 mg/L ProAV as compared to that registered in the

246 absence of the additives. Similar experiments with ROS-interceptors have been

247 formerly employed to confirm/discard the participation of O2(1g), O2– , H2O2 and

248 OHrespectively in a given oxidative event.35,36,37 The enzyme SOD dismutates the

249 species O2– (reaction (18)), whereas CAT decomposes H2O2 (reaction (19)),

250 mannitol deactivates the species OH(reaction(20)) and NaN3 physically quenches

251 O2(1g) (reaction (16)), Scheme 1, with NaN3 instead of Q.

252 2 O2– + 2 H+ + SO → O2(3Σg-) + H2O2 (18)

253 2 H2O2 + C T → 2 2O + O2(3Σg-) (19)

254 OH+ mannitol → eactivation (20)

255 Please insert Figure 5 here

256 3.5 Photoprotective effect of diOHMeCh towards tryptophan oxidation.

257 The eventual protective effect of diOHMeCh on the ROS-mediated oxidation of

258 proteins, the Rf- sensitized photooxidation of the amino acid tryptophan (trp) was

259 employed.29
260 The rates of oxygen consumption by 0.5 mM trpr and 0.5 mM diOHMeCh and the

261 mixture of both substrates in MeOH were determined. The rates were taken as a

262 measure of the global photooxidative progress by monitoring up to 15% conversion

263 of the starting material. In relative terms, the obtained rate values were 1; 0.31 and

264 0.37 respectively. This means that the photodegradation of trp, in the presence of

265 diOHMeCh suffers a decrease of ca.63% as compared to that in that absence of

266 the polyphenol.

267 It is well known that trp is photooxidizable upon Rf-sensitization.38 The degradative

268 process operates through a combination of radical-mediated and O2(1g)- mediated

269 mechanisms. Garcia et al.38 reported on the generation of O2–; H2O2; OH and

270 O2(1g) in the Rf-sensitized photodegradation of trp, and we determined a kr = 3.5

271 107 M-1s-1for reaction (17) with the amino acid instead of Q.39

272

273 4. DISCUSSION

274 4.1 Overall antioxidant activity of ProAV

275 DPPH radical scavenging action and antioxidant activity towards linoleic acid

276 clearly demonstrate the overall defensive power of ProAV against aggressive

277 deleterious agents.27,28 The antioxidative action of ProAV is accompanied by its

278 concomitant degradation. This fact mainly involve an exclusive O2(1g)-mediated

279 pathway in the case of RB-sensitization and a group of radical-driven reactions in

280 addition to the mentioned O2(1g) degradation in the case of Rf-sensitization,

281 respectively. Nevertheless the ProAV fading is kinetically moderate and less than

282 two fold higher than the rate of fading experimented by trolox (Figure 2), a
283 recognized artificial antioxidant.29

284 4.2 The promotion and quenching of ROS by ProAV

285 ProAV reacts with O2(1g), O2–, H2O2 and OH. The species H2O2 and OH• are not

286 spontaneously formed during the Rf photoirradiation. Hence, the initial action of

287 ProAV could be considered as a promotion towards these ROS generation. The

288 operation of this possible pathway has been demonstrated for a series of electron-

289 donors, including several flavonoids, in the presence of photoirradiated Rf.30

290 The radical reaction initially produces RfH and ProAV +(reaction 8). The latter

291 species, representing one or more electron-donating components of the propolis,

292 possibly decays to products and/or reacts with ROS generated in the medium. 3Rf*

293 could be quenched either by O2(3Σg-) (reaction (14)) or by ProAV (reaction (8)).

294 Whether one of these processes is kinetically dominant depends on the respective

295 concentration of the quenchers and on the rate constant values for reactions (8)

296 and (14). There are several reports in the literature with different flavonoids acting

297 as efficient electron donors towards 3Rf*, a species that in a simultaneous event

298 can produce O2(1g).40 The fact that under identical experimental conditions the

299 detection tests for O2(1g) on one hand and O2–, H2O2 and OH on the other,

300 clearly indicates that the pathways (8) and (14) are kinetically competitive in some

301 extent, under the employed experimental conditions.

302 4.3 Photoprotection of trp-photooxidation by diOHCh

303 The dominant physical scavenging of O2(1g), represents a positive result arising

304 in the context of oxidative interactions between biomolecules. In this case the

305 considerably low values for the kr/kt quotient exhibited diOHCh and diOHMeCh
306 is a desirable quality. The final result is the elimination of O2(1g) without

307 appreciable loss of the scavenger.

308 This action constitutes a protection for proteins, DNA and other cell matrix

309 components.41 This photoprotective effect exerted diOHCh was evident as

310 observed by the clear decrease in the rate of oxygen uptake of the system trp +

311 chalcone as compared to that of the amino acid alone, upon Rf- sensitization.

312

313 References
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315 2. Hudson BJ. Food Antioxidants. Elsevier Applied Food Science. London; 1990.
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354 20. Montaña MP, Pappano N, Debattista N, Ávila V, Posadaz A, Bertolotti SG, et al.
355 The activity of 3- and 7-hydroxyflavones as scavengers of superoxide radical anion
356 generated from photo-excited riboflavin,Can. J Chem 2003;81:909–914.
357 21. Montaña MP, Massad WA, Criado S, Biasutti A, García NA. Stability of flavonoids
358 in the presence ofriboflavin-photogenerated reactive oxygen species. A kinetic and
359 mechanistic study on quercetin, morin and rutin. Photochem Photobiol 2010;86:827–
360 834.
361 22. Agüero MB, González M, Lima B, Svetaz L, Sánchez L, Zacchino S, et al.
362 Argentinean Propolis from Zuccagnia punctata Cav. (Caesalpinieae) Exudates:
363 Phytochemical Characterization and Antifungal Activity, J Agric Food Chem
364 2010;58:194–201.
365 23. Szliszka E, Czuba ZP, Domino M, Mazur B, Zydowicz G, Krol W. Ethanolic
366 Extract of Propolis (EEP) enhances the apoptosis- inducing potential of TRAIL in
367 cancer cells. Molecules 2009;14:738–754.
368 24. Kumazawa S, Hamasaka T, Nakayama T. Antioxidant activity of propolis of
369 various geographic origins. Food Chem 2004;84:329–339.
370 25. Lima B, Tapia AA, Luna L, Fabani MP, Schmeda-Hirschmann G, Podio NS, et al.
371 Main Flavonoids, DPPH Activity, and Metal Content Allow Determination of the
372 Geographical Origin of Propolis from the Province of San Juan (Argentina), J Agric
373 Food Chem 2009;57:2691–2698.
374 26. Ahn MR, Kumazawa S, Usui Y, Nakamura J, Matsuka M, Zhu F, et al.
375 Antioxidant activity and constituents of propolis collected in various areas of China.
376 Food Chem 2007;101:1383–1392.
377 27.Tratniek PG, Hoigné J. Oxidation of substituted phenols in the environment: A
378 QSAR analysis of rate constants for reaction with singlet oxygen. Environ Sci Technol
379 1991;25:1596–1604.
380 28. Criado S, Allevi C, Ceballos C, García NA. Visible-light-promoted degradation of
381 the commercial antioxidants butylated hydroxyanisole (BHA) and butylated
382 hydroxytoluene (BHT). A kinetic study, Redox Report 2007;12:282–288.
383 29. Wilkinson F, Helman WP, Ross A. Rate constants for the decay of the lowest
384 electronically excited singlet state of molecular oxygen in solution. An expanded and
385 revised compliation. J Phys Chem Ref Data 1995;24:663–1021.
386 30. Amat-Guerri F, García NA. Photodegradation of hydroxylated N-heteroaromatic
387 derivativesin natural-like aquatic environments.A review of kinetic data of pesticide
388 model compounds. Chemosphere 2005;59:1067–1082.
389 31. Fritz BJ, Matsui K, Kasai S, Yoshimura A. Triplet lifetime of some flavins,
390 Photochem Photobiol 1987;45:539–541.
391 32. Gutiérrez S, Criado S, Bertolotti S, García NA. Dark and photoinduced
392 interactions between trolox, a polar-solvent-soluble model for vitamin E, and
393 riboflavin. J Photochem Photobiol B 2001;62:133–139.
394 33. Wilkinson F, Helman WP, Ross AB. Quantum yields for the photosensitized
395 formation of the lowest electronically excited singlet state of molecular oxygen in
396 solution. J Phys Chem Ref Data 1993; 22:133–262.
397 34. García NA. Singlet molecular oxygen-mediated photodegradation of aquatic
398 phenolic pullulants. A kinetic anf mechanistic overview. J Photochem Photobiol B:
399 Biology 1994;22:185–196.
400 35. Escalada JP, Pajares A, Gianotti J, Massad W, Bertolotti S, Amat-Guerri F, et al.
401 Dye-sensitized photodegradation of the fungicide carbendazim and related
402 benzimidazoles. Chemosphere 2006;65:237–244.
403 36. Silva E, Herrera L, Edwards AM, De La Fuente J, Lissi E. Enhancement of
404 riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by uronic
405 acid. Photochem Photobiol 2005;81:206–211.
406 37. Silva E, Edwards AM, Pacheco D. Visible light-induced photooxidation of glucose
407 sensitized by riboflavin. J Nutr Biochem 1999;10:181–185.
408 38. Garcia J, Silva E. Flavin-sensitized photooxidation of amino acids present in a
409 parenteral nutrition infusate: protection by ascorbic acid. J Nutr Biochem 1997;8:341–
410 5.
411 39. Posadaz A, Biasutti A, Casale C, Sanz J, Amat-Guerriand F, García NA. Rose
412 Bengal-Sensitized Photooxidation of the Dipeptides Trp-Phe, Trp-Tyr and Trp-Trp.
413 Kinetics, Mechanism and Photoproducts. Photochem Photobiol 2004;80:132–138.
414 40. Montaña P, Pappano N, Debattista N, Ávila V, Posadaz A, Bertolotti SG, et al.
415 The activity of 3- and 7-hydroxyflavones as scavengers of superoxide radical anion
416 generated from photo-excited riboflavin. Can J Chem 2003;81:909–914.
417 41. Spikes JD, Shen HR, Kopeccková P, Kopecek J. Photodynamic crosslinking of
418 proteins. III. Kinetics of the FMN- and rose bengal-sensitized photooxidation and
419 intermolecular crosslinking of model tyrosine-containing N-(2-Hydroxypropyl)
420 methacrylamide copolymers. Photochem Photobiol 1999;70:130–137.
421
Non-colour figure
Click here to download Non-colour figure: Scheme and Figure captions.docx

Scheme and Figure captions

Scheme 1. Possible reaction pathways in a Riboflavin-photosensitized process.

Figure 1. Panel A: % DPPH radical scavenging activity by catechin (●) and
ProAV(□) as a function of substrate concentration. See details in the experimental
section. Panel B: % of Absorbance decrease at 470 nm a function of incubation
time of a solution of β-carotene/linoleic acid in the absence (●) and in the presence
of BHT (▲) and ProAV (■). See details in the experimental section.

Figure 2.Changes in the UV-Vis absorption spectrum of Rf A446 = 0.4 plus 57μg/ml
ProAV in MeOH-H2O (1:1;V/V), taken vs. A446 = 0.4, upon visible-light irradiation.
Inset A: Oxygen consumption vs. photoirradiation time in MeOH-H2O (1:1;V/V) for
the systems: Rf A446 = 0.40 plus (a) 38μg/ml ProAV and (b) 38μg/ml trolox. Inset
B: Changes in the UV-Vis absorption spectrum of RB (A630 = 0.4) plus 64μg/ml
ProAV taken vs. RB (A630 = 0.4) upon visible-light irradiation in MeOH. In all cases
cut-off at 430 nm.
Figure 3. Stern-Volmer plot for the quenching of 1Rf* by ProAV. I0 and I represent
the stationary fluorescence intensities of Rf (A446 = 0.1) in the absence and in the
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 photoirradiation at irradiating with 44510 nm in
argon-saturated methanolic solution in the absence (1 in the abscise axis) and in
the presence 15 μg/ml ProAV; 36 μg/ml ProAV and 60 μg/ml ProAV (2; 3; and 4 in
the abscise axis respectively).
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 O 2(1Δg) in the absence
(a) and in the presence (b) of 68 μg/ml ProAV in MeOD. Photosensitizer: RB (A532
= 0.18).
Figure 5.Bars diagram for relative rate values of oxygen uptake by the system Rf
(A446 = 0.4) plus 38 mg/L ProAV as a function of photoirradiation time (cut-off 430
nm), in MeOH-H2O (1:1, v/v) solutions in the absence (1) and in the presence of
10μg/ml CAT (2); 10μg/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.
Non-colour figure
Click here to download Non-colour figure: Scheme 1.doc

h
Rf 1Rf* 3Rf* (5)
1Rf* + Q Rf + Q (6)
1k
q
3Rf* + O2(3-g) Rf•+ + O2 •- (7)
keT
3Rf* + Q 3k Rf•- + Q •+ (8)
q

Rf •- + H+ RfH • (9)

2 RfH • RfH2 + Rf (10)

RfH2 + O2(3-g) [Rf H2•+ + O2 •- ] Rf + H2O2 (11)

H2O2 + O2 •- O2 + OH • + OH- (12)

O2 •- + Q Products 13 (13)

OH • + Q Products 14 (14)

H2O2 + Q Products 15 (15)

3Rf* + O2(3-g) Rf + O2 (1g) (16)

kET
O2 (1g) kd O2(3-g) (17)

O2 (1g) + Q kq O2(3-g) + Q (18)

O2 (1g) + Q kr Products 19 (19)

Scheme 1: Possible reaction pathways in a Riboflavin-photosensitized process

Non-colour figure
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Non-colour figure
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Non-colour figure
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Copyright Form
Manuscript (.doc/.docx)
Click here to download Manuscript (.doc/.docx): Propolis. Corrected Ms.docx

1 The activity of propolis in the scavenging of Vitamin B2-

2 photogenerated ROS

5 Mariela González1*, María L. Tereschuk1*, Susana Criado3, Eugenia Reynoso3,

6 Cecilia Challier3, María Belén Agüero4, Lorena Luna4, Gabriela Ferrrari2,

7 María P. Montaña2 and Norman A. García3

1
8 Departamento de Ingeniería de Procesos y Gestión Industrial. Facultad de
9 Ciencias Exactas y Tecnología. Univ. Nac. de Tucumán, Av. Independencia 1800,
10 (4000) Tucumán. Argentina.
2
11 Área de Química-Física, Univ. Nac. de San Luis, Lavalle 1151, (5700) San Luis.
12 Argentina.
3
13 Departamento de Química, Univ. Nac. de Río Cuarto, Campus Universitario,
14 (5800) Río Cuarto. Argentina
4
15 Instituto de Biotecnología. Inst. de Cs. Básicas, Universidad Nacional de San
16 Juan, Av. Libertador General San Martín 1109 (O), 5400 San Juan. Argentina.
17
18

19

20

21

22

23

24

25

26 (*) Corresponding author. MG: magonzalez@herrera.unt.edu.ar; MLT:

27 mtereschuk@herrera.unt.edu.ar; Tel.: +54 381 4364093.

28

29 Abstract
30 Objectives: The study was focused on the activity of propolis from Amaicha del
31 Valle, Argentina (ProAV) as a promoter and scavenger of Riboflavin (Rf) -
32 photogenerated reactive oxygen species (ROS).
33 Methods: Through a kinetic and mechanistic study, employing stationary and time-
34 resolved photochemical and electrochemical techniques, the protecting activity of
35 ProAV was investigated.
36 Results: In the absence of light and Rf, ProAV exerted a relatively efficient
37 inhibitory effect on DPPH radicals and acts as a protector of artificially-promoted
38 linoleic acid oxidation. Under aerobic visible-light-irradiation conditions, in the
39 presence of Rf as the only light-absorber species, a complex picture of competitive
40 processes takes place, starting with the quenching of singlet and triplet
41 electronically excited states of Rf by ProAV. The species O2( g), O2.- , H2O2 and
1

42 OH. are generated and interact with ProAV.

43 Discussion: ProAV behaves as an efficient ROS scavenger. It is scarcely
44 photooxidized by interaction with the mentioned ROS. Quantitative results indicate
45 that ProAV is even more resistant to photooxidation than the recognized
46 antioxidant trolox. Two dihydroxychalcones, mostly present in the ProAV
47 composition, are responsible for the protecting activity of the propolis.
48
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69 1. INTRODUCTION
70 A wide variety of aggressive agents such as free radicals and reactive oxygen

71 species (ROS) can be generated in the human body and in different kind of foods.

72 Some defences play a protective role and most of these defences are antioxidants.

73 It means compounds that inhibit or delay the oxidation of other molecules by

74 preventing the initiation or propagation of oxidizing chain reactions. There are two

75 basic categories of antioxidants, namely synthetic and natural. In general, both

76 categories are formed by compounds containing phenolic structures.1-3 Synthetic

77 antioxidants such as butylatedhydroxyanisole (BHA) and butylatedhydroxytoluene

78 (BHT) have been employed since the beginning of 1900. However, there are

79 restrictions on the use of these compounds, in connection to human health.4,5

80 Thus, the interest in natural antioxidants has increased considerably the research

81 activity on different types of native substrates.6,7

82 Apis bees prepare a product named propolis to fulfil holes and to embalm dead

83 predators inside the hives. This product is elaborated from plant resins, bee wax

84 and secretions of the head glands of workers bees. Propolis have shown biological

85 activity which has introduced it as a potential active principle for pharmaceutical

86 preparations.8-10

87 Regarding the chemical composition of propolis, it must be said that it contains a

88 series of plant metabolites, including volatiles, that are produced by different plant
11
89 species and are not the same all over the world . Moreover, it was revealed that
 90 propolis samples from different geographic and climatic regions, always presented
12
91 biological activity, although their chemical composition was completely different.

92 The functional properties are mainly ascribed to a few substances, including

93 flavonoids, such as flavones and chalcones, and a variety of phenolic acids.13,14

94 Most research work has been devoted to the antioxidant activity of flavonoids,

95 which is due to their ability to reduce the free radical formation and to scavenge

96 oxidative agents.15 Riboflavin (Rf), is one of the most important natural visible-light

97 absorbers present in a wide variety of live systems and foods. Both beneficial and

98 noxious effects of this vitamin under dark and photoirradiation conditions have

99 been addressed in recent reports.16,17,18 Rf has been postulated as a sensitizer

100 for photopromoted reactions –mainly photooxidations– that can produce

101 physiological changes in the surrounding molecules.19-21. It is well known that Rf

102 and derivatives generate the ROS singlet molecular oxygen (O2(1g)) and

103 superoxide radical anion (O2•─) after adequate photoirradiation.22,23,24 In the

104 presence of an electron donor also the photogeneration of the ROS hydrogen

105 peroxide (H2O2) and hydroxy radical (OH•) has been reported. 25,26.

106 In this work we discuss results of a systematic kinetic and mechanistic study on the

107 potential reactive interactions of Rf-photogenerated ROS with ProAV and with two

108 chalcones present in it. Thus we evaluated both the activity of ProAV in the

109 promotion and scavenging of Rf-photogenerated ROS and the stability of propolis

110 in the presence of the oxidative species.

111 2. EXPERIMENTAL

112 2.1 Materials

113 A 30 g of propolis sample (ProAV) was collected from beehives situated in

114 Amaicha del Valle (Tucumán, Argentina). It was stored at – 18 ºC.

115 Riboflavin (Rf), superoxide dismutase (SOD) from boverythrocytes, catalase from

116 bovine liver, mannitol, L-tryptophan, β – carotene, Folin - Ciocalteu’s phenol

117 reagent, and Mono deuterated MeOH (MeOD) were purchased from Sigma Chem.

118 Co. Rose Bengal (RB), Sodium azide (NaN3) and Furfuryl alcohol (FFA),were

119 provided by Aldrich, Merck and Riedel de Haën respectively. The radical 1,1-

120 Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Aldrich. Methanol (MeOH)

121 HPLC quality, was purchased from Sintorgan. MeOD was employed in the time-

122 resolved determinations of O2(1g) phosphorescence in order to enlarge the lifetime

123 of this species. Water was triply distilled. Ethanolic Extract of propolis ProAV (EE)

124 was obtained as described below and 2’,4’- dihydroxychalcone (diOHCh) and
27
125 2’,4’- dihydroxy-3’-methoxychalcone (diOHMeCh) were isolated at the

126 Laboratory of Dr. Alejandro Tapia from the University of San Juan (UNSJ), San

127 Juan, Argentina.

128 2.2 Methods

129 2.2.1 Preparation of Propolis EE and isolation of diOHCh and diOHMeCh

130 Preparation of EE was performed according to Szliszka et al.28 The extraction yield

131 was 69%. Isolation of Chalcones diOHCh and diOHMeCh from Zuccagnia punctata

132 Cav. collected in Amaicha del Valle, was performed according to methods of

133 Agüero et al.27

134 2.2.2 Total Phenolic compounds

135 Total polyphenol contents in EE were determined by the Folin-Ciocalteau

136 colorimetric method, according to Kumazawa et al.29 EE solution was mixed with

137 Folin-Ciocalteau reagent and 0.5 mL of 10% Na 2CO3. EE were evaluated at the

138 final concentration of 20 μg/mL. Total polyphenol content was expressed as grams

139 gallic acid equivalent/ 100 gram of sample.

140 2.2.3 Free Radical Scavenging Activity on DPPH

141 Free radical scavenging effects of ProAV and catechin as reference were

142 assessed by the fade of a ethanolic solution of 1,1-diphenyl-2-picrylhydrazyl radical

143 as previously reported by Lima et al.30 EE was assayed at concentrations of 100

144 μg/mL. Fade percentage was calculated as follows (Eq. (1)):

145 (1)

146 The loss of color, spectrophotometrically followed at 517 nm, indicated the free

147 radical scavenging efficiency.

148 2.2.4 Antioxidant activity on linoleic acid oxidation

31
149 This analysis was carried out using the method developed by Ahn et al. with

150 some modifications. We dissolved 5 mg of β-carotene in 10 mL of chloroform, 134

151 µL of linoleic acid and 380 µL of Tween 20 was added. Test samples were

152 evaluated at the final concentration of 90 μg/mL and BHT were used as the

153 reference. The antioxidant activity % was expressed relative to the initial

154 absorbance of the sample during 300 min incubation using eq. (2):

155 (2)
156 AA % = antioxidant activity percent

157 Ao = Initial Absorbance at 470 nm and t = 0

158 At = Absorbance at measurement time at 470 nm (intervals of 60 min during 300

159 min).

160 2.2.5 Absorption and fluorescence experiments

161 Ground state absorption spectra were registered employing a Hewlett Packard

162 8453 diode array spectrophotometer. For the stationary Rf fluorescence

163 experiments, a RF 5301-PC Shimadzu spectrofluorimeter was used. Excitation

164 and emission wavelengths were 446 and 515 nm, respectively.

165 A classical Stern-Volmer treatment of the data was applied through Eq. (3), where

166 I0 and I are the respective fluorescence intensities of Rf in the presence and in the

167 absence of the eventual quencher Q, and Ksv (Ksv= 1kq x 10) is the Stern-Volmer

168 constant for the quenching of excited singlet Rf (1Rf*), being 1kq (see Scheme 1)

169 and 10 the respective rate constants for the dynamic quenching of 1Rf* and the

170 lifetime of 1Rf*.

171 I0 / I= 1 + Ksv [Q] (3)

172 2.2.6 Time resolved O2(1g) phosphorescence detection (TRPD)

173 The total quenching rate constant (kt, see Scheme 1) for O2(1g) deactivation by an

174 eventual quencher Q, was determined by near-IR time resolved phosphorescence,

175 employing eq. (4), being  and 0 the respective O2(1g) lifetimes in the presence

176 and in the absence of Q.

177 0 /  = 1+ kt 0 [Q] (4)

178 The 532 nm output from a Nd:Yag laser (Spectron) was used as the excitation

179 source. The emitted (O2(1g)) phosphorescence at 1270 nm was detected at right

180 angles using a Edinburgh EI-P Germanium detector, after having passed through

181 1270 nm-interference and two wratten filters. The output of the detector was

182 coupled to a 400 MHz digital oscilloscope (HP 54504A) and to a personal

183 computer for signal processing. Air equilibrated solutions were employed in all

184 cases.

185 2.2.7 Stationary photolysis and oxygen uptake experiments

186 Stationary aerobic photolysis of aqueous solutions containing ProAV plus either Rf

187 or RB were carried out in a PTI unit, provided with a high pass monochromator and

188 150 W Xe lamp, irradiating with 44510 nm, or in a home-made photolyser for 5

189 non-monochromatic irradiation (150 W quartz-halogen lamp). In this case cut-off

190 filters (430 nm).

191 The Rf- or RB-sensitized photooxygenation rates of ProAV, diOHCh or diOHMeCh

192 were determined by evaluation of the initial slopes of oxygen consumption vs.

193 irradiation time, employing a specific oxygen electrode (Orion 97–08).

194 Reactive (chemical) rate constants for the interaction Q-O2(1Δg) (kr, see Scheme 2)

195 were obtained as described by Tratniek and Hoigné32, from the ratio of the first

196 order slopes of B6D and reference consumption, each at the same concentration,

197 yielding kr/krR. The reference was FFA, with a krR =1.2 x 108 M–1s–1.
198 The anaerobic rates of Rf photodecomposition were determined in the absence

199 and in the presence of different ProAv concentrations by the decrease in the 446

200 nm absorption band of the vitamin upon 5 min irradiation with visible light (cut off

201 430) employing MeOH as a solvent, in Ar-bubbled solutions.

202 3. RESULTS

203 3.1 ProAV overall antioxidative activity.

204 Comparative runs of the radical scavenger action on DPPH by ProAV and by the
33
205 well-known radical trapper catechin were performed . Results are shown in Figure

206 1A.

207 Please, insert Figures 1A and 1B here.

208 Next, in Figure 1B are shown comparative runs of the antioxidant activity exerted

209 by ProAV and by the recognized antioxidant BHT, on linoleic acid oxidation. BHT is

210 one of the artificial compounds most extensively employed to prevent oxidant
34
211 rancidity in numerous commercial products. From both experiments in Figure 1

212 it can be concluded that ProAV unambiguously show antioxidant capacity. The

213 mentioned initial results merely constitute a coarse evaluation of ProAV as an

214 antioxidant, in the absence of any photopromoted process. Neverhteless, these

215 assays strongly suggest the potentiality of the substrate as an overall defensive

216 agent against aggressive oxidative species.

217 On this basis, the interaction of ProAV with photochemically generated ROS in the

218 absence and in the presence of oxidizable targets constitutes a topic of research

219 interest. Hence, we decided to go deeper in the properties of ProAV as an

220 antioxidant agent, in order to know more on:

221 a) The fate of ProAV in the presence of photogenerated ROS.

222 b) Kinetic and mechanistic aspects governing of the potential antioxidant

223 activity of ProAV.

224 c) The antioxidant capacity of ProAv as a photoprotector of biological targets.

225 As mentioned, the natural compound Rf was employed in order to photogenerate

226 ROS.

227 The Rf (A446 = 0.4)–sensitized photoirradiation of air-equilibrated solutions of ProAV

228 (57μg/ml) in MeOH-H2O (1:1;V/V) produces different changes in the absorption

229 spectrum of ProAV. The irradiation wavelength was higher than 430 nm, a spectral

230 region where only Rf absorbs the incident light. Figure 2 shows the absorption

231 spectrum of the mixture (Rf plus ProAV vs. Rf) at different irradiation times.

232 Please insert Figure 2 here

233 Parallel photoirradiation of a similar solution to the above described one (with 38

234 μg/ml ProAV in this case) gave rise to oxygen consumption. No oxygen uptake was

235 observed in the absence of light or ProAV. In order to standardize the evaluation of

236 the rate of oxygen uptake, the same described experiment was performed by

237 changing ProAV by and identical W/W concentration of the well-known artificial

238 antioxidant Trolox.35 The phenolic derivative is considered a water soluble model

239 compound for vitamin E. 36

240 Results shown in Figure 2 inset indicate that the rate of oxygen consumption by

241 ProAV under Rf-photosensitization is only 65% of that corresponding to trolox.

242 From the above-mentioned experimental evidence, ProAV appears as a potential

243 interceptor of Rf-photogenerated oxidative species and three particularly relevant

244 observations arise: a) ProAV is chemically affected by the visible light-irradiation in

245 the presence of Rf as a dye-sensitizer; b) oxygenated species participate in the

246 process and c) electronically excited states of Rf seems to participate in the overall

247 interaction.

248 Individualization of the active excited states of the sensitizer, optimal sensitizing

249 conditions, oxygen requirement and local concentration necessary for the effective

250 interaction of the involved reaction partners may help to elucidate and interpret the

251 reaction mechanism. This point was discussed on the basis of the following well

252 known reaction sequence (Scheme 1).37 Q represents an electron donor species,

253 namely ProAV in most of the experiments of the present contribution. In some

254 cases the propolis was replaced by diOHCh or diOHMeCh, in order to

255 comparatively evaluate the behaviour of these substrates. Both flavonoids are the

256 most abundant antioxidant components detected in the ProAV sample.

257

258 Please, insert Scheme 1 here

259 According to Scheme 1, the absorption of incident light promotes Rf to

260 electronically excited singlet (1Rf*) and triplet (3Rf*) states, (5). Both states can be

261 quenched by Q through reactions (6) and (8). The latter by means of an electron

262 transfer process by which the respective semireduced and semioxidized forms, Rf •-

263 + Q•+, are produced. The species Rf•- is rapidly protonated (9). Reactions (10) and

264 (11) represent the reduction of the neutral Rf radical and generation of the ROS

265 O2•- and (H2O2) respectively. By means of reaction (12) the radical OH• is produced.

266 Reactions (13); (14) and (15) indicate oxidative processes of Q mediated by the
267 species O2•- ,
OH• and (H2O2) respectively. The energy transfer reaction (16)

268 generates O2(1g) with a reported quantum yield of 0.49 in MeOH (reaction (14),
38,39
269 which can decay by interaction with solvent molecules (17), or interact with Q

270 in a mere physical fashion (18) or in a reactive fashion (19). An overall rate constant

271 kt for O2(1g) quenching is defined as the addition of the rate constants kq and kr

272 (processes (18) and (19) respectively).

273 3.2 Quenching of 1Rf*and 3Rf*by ProAV

274 Rf presents an intense fluorescence emission band, centered at 515 nm. The
19
275 reported emission quantum yield (ΦF) is 0.25. In the presence of ProAV, the

276 fluorescence quenching of 1Rf* produces a decrease in the stationary emission

277 intensity, but the shape of the fluorescence spectrum does not change, as shown

278 in Figure 3, inset A. Figure 3, main panel, shows the quenching plot from which a

279 Stern-Volmer constant of 7 x 10-4 L/mg. s-1 was obtained in MeOH, employing Rf

280 (A446 = 0.1) . The reported value for the fluorescence lifetime of the vitamin in

281 MeOH is 2 ns.22

282 Please, insert Figure 3 here

283 It is well known that the anaerobic photodegradation of Rf under visible light

284 irradiation predominantly proceeds through the triplet state, and the rate of the
19.
285 process can be estimated by absorption spectroscopy Comparative irradiations

286 of Ar-saturated solutions of Rf in MeOH under identical experimental conditions, in

287 the absence and in the presence of different ProAV concentrations showed that

288 this rate decreased in the presence of ProAV in the range 0-60 g/mL (Figure 3,
289 inset B). At the said ProAV concentrations, practically no fluorescence quenching

290 of Rf occurs. A simple calculation employing the Stern-Volmer constant for the

291 fluorescence quenching, indicates that an inhibition of ca. 7% in the emission of

292 Rf* should be expected for a ProAV concentration of 60 g/mL. In parallel, a

1

293 decrease of ca. 78% in the degradation rate of Rf was observed for this

294 concentration of ProAV (Figure 3, inset B). On this basis, the experimental data

295 strongly supports the idea that a long-lived triplet state, intermediate in the

296 photolysis of Rf, can be efficiently quenched by relatively low ProAV

297 concentrations. The reported lifetime for 3Rf* in MeOH is 12 s. 19

298 3.3 Quenching of O2(1g) by ProAV and diOHCh

299 The eventual interaction of ProAV and diOHCh with O2(1g) was explored

300 employing RB as a dye sensitizer. The xanthene dye generates O2(1g) in an

301 exclusive fashion with a quantum yield of ca. 0.7 in MeOH. This procedure avoids

302 the eventual generation of other ROS such as O2•- , H2O2 and OH•, predictable in

303 a Rf-sensitized process. 40

304 The RB- sensitized photoirradiation of ProAV (RB (A630 = 0.4)) in MeOH produced

305 evident changes in the absorption spectra of the propolis shown in Figure 2, inset B.

306 Besides, the decrease of the emissive lifetime of O2(1g) in the presence of 68

307 mg/L ProAV in MeOD (Figure 4, inset) unambiguously demonstrates the quenching

308 of O2(1g) by the propolis with the oxidative species. Rate constant values kt = 3.4

309 x 107 M-1s-1 and 2.8 x 107 M-1s-1 were obtained for diOHCh and diOHMeCh in
310 MeOD. It is in the same order of magnitude of reported kt values for other

311 flavonoids.40 The kr value was evaluated by comparison with the well-known

312 reference compound FFA. No oxygen uptake by the systems 0.5 mM diOHCh or

313 diOHMeCh plus RB (A630 = 0.4) in MeOH was observed. Hence a kr value ˂ 10-6

314 M-1s-1 can be ascribed for both of them a. The kr/kt ratio, which indicates the

315 fraction of overall quenching of O2(1Δg) by the substrate that effectively leads to a

316 chemical transformation indicates that the flavonoids are exclusive physical

317 quenchers of the oxidative species.

318 A rate constant value of 1.4 x 108 M-1s-1 was obtained for diOHCh in the presence

319 of 10 mM NaOH. The observed increase in the kt value, as compared to that

320 obtained in the absence of alkali, is a well known effect in phenolic derivatives.

321 Under these conditions the phenolic hydroxyl groups are ionized, increasing

322 significantly their electron transfer ability towards O2(1Δg). 41

Experimental kinetic

323 data for the interaction diOHCh-O2(1Δg) is shown in Figure 4, main panel.

324 Please insert Figure 4 here

325 3.4 The interaction ProAV and diOHCh with ROS

326 The distinction between radical-mediated and singlet oxygen mediated oxidative

327 processes is a key piece in determining the driving mechanistic in a given event.

328 One interesting work in this area belongs to Sardar et al. The authors identify the
42
329 source of ROS photogeneration in a complex system through indirect evidence.

330 In order to evaluate the nature of the interaction of Rf-photogenerated ROS with

331 ProAV oxygen consumption experiments in the presence of specific ROS

332 interceptors were carried out. The presence of 10 mM NaN3; 1 μg/ml SOD; 1 μg/ml
333 CAT and 10 mM mannitol decreases the rate of oxygen uptake of MeOH-H2O (1:1,

334 v/v) solutions containing Rf (A446 = 0.4) plus 38 mg/L ProAV as compared to that

335 registered in the absence of the additives. Similar experiments with ROS-

336 interceptors have been formerly employed to confirm/discard the participation of

337 O2(1g), O2– , H2O2 and OHrespectively in a given oxidative event.43,44,45 The

338 enzyme SOD dismutates the species O2– (reaction (20)), whereas CAT

339 decomposes H2O2 (reaction (21)), mannitol deactivates the species OH (reaction

340 (22)) and NaN3 physically quenches O2(1g) (reaction (18)), Scheme 1, with NaN3

341 instead of Q.

342 2 O2– + 2 H+ + SOD → O2(3Σg-) + H2O2 (20)

343 2 H2O2 + CAT → 2 H2O + O2(3Σg-) (21)

344 OH+ mannitol → deactivation (22)

345 Please insert Figure 5 here

346 3.5 Photoprotective effect of diOHMeCh towards tryptophan oxidation.

347 In order to evaluate the eventual protective effect of diOHMeCh on the ROS-

348 mediated oxidation of proteins, the Rf- sensitized photooxidation of the amino acid

349 tryptophan (trp) was employed.35

350 The rates of oxygen consumption by individual solutions of 0.5 mM trpr, 0.5 mM

351 diOHMeCh and 0.5 mM trpr plus 0.5 mM diOHMeCh were determined in MeOH.

352 The rates were taken as a measure of the photooxidative degradation of the amino

353 acid, by monitoring up to 15% of oxygen uptake. In relative terms, the obtained rate
354 values were 1; 0.31 and 0.37 respectively. This means that the rate of trp

355 photodegradation undergo a decrease of ca.63% in the presence of diOHMeCh, as

356 compared to the rate value in the absence of the polyphenol.

357 It is well known that trp is photooxidizable upon Rf-sensitization.46 The degradative

358 process operates through a combination of radical-mediated and O2(1g)- mediated

359 mechanisms. Garcia et al.46 reported on the generation of O2–; H2O2; OH and

360 O2(1g) in the Rf-sensitized photodegradation of trp, and a kr value of 3.5 107 M-
1 -1
361 s was determined for reaction (19) with trp instead of Q.47

362

363 4. DISCUSSION

364 4.1 Overall antioxidant activity of ProAV

365 Preliminary assays on DPPH radical scavenging action and the protective activity

366 towards linoleic acid, clearly demonstrate an overall anti-oxidative tendency of

367 ProAV.32,34

368 The systematic study employing RB and Rf as photosensitizers allows the

369 identification of mechanistic pathways involving ProAVas as both an efficient

370 O2(1g) quencher in the case of sensitization by the xanthene dye and as a

371 scavenger of several ROS, in the case of Rf-sensitization.

372 The antioxidative action of ProAV is accompanied by its concomitant degradation.

373 Nevertheless ProAV fading is small in kinetic terms. A quantitative evaluation

374 shown in Figure 2 establishes that the photooxidation rate of ProAV reaches only

375 65% of the value obtained for the stable and widely employed artificial antioxidant

376 trolox. 35
For the case of the interaction trolox-O2 (1g), a value of kr/kt ~ 0.1 has
377 been reported by ourselves in alcoholic solvent mixtures, demonstrating the

378 efficiency of the phenolic compound as a physical scavenger of the oxidative

379 species. 36

380 4.2 The promotion and quenching of ROS by ProAV

381 ProAV reacts with O2(1g), O2–, H2O2 and OH. The species H2O2 and OH• are not

382 spontaneously formed during the Rf photoirradiation. Hence, the initial action of

383 ProAV could be considered as a promotion towards these ROS generation. This

384 effect has been demonstrated for a series of electron-donors, including several

385 flavonoids, in the presence of photoirradiated Rf.37 In short, reaction (8) produces

386 RfH- and ProAV +. The latter species, can decay to products and/or react with

387 ROS generated in the medium, whereas the species Rf radical cation may initiate

388 the series of reactions (9)-(12) producing H2O2 and OH and additional O2–.

389 According to Scheme 1, 3Rf* can be quenched either by O2(3Σg-) (reaction (16)) or

390 by ProAV (reaction (8)). Whether one of these processes is kinetically dominant

391 depends on both the respective concentration of the quenchers and the rate

392 constant values for reactions (8) and (16). Several research works indicate that

393 flavonoids –the main components of ProAV– may act as efficient electron-donors

394 towards 3Rf*. Rate constant values for process (8) in the order of 109 M-1s-1 have
48.
395 been reported On the other hand, the reported rate constant value in MeOH for

396 process (16), the energy transfer step responsible for O2(1g) generation, is 1.2 x

397 109 M-1s-1.49

398 All this data indicates that for the same concentrations of dissolved oxygen and

399 ProAV, processes (8) and (16), are kinetically competitive. This point is reinforced
400 by the fact that under identical experimental conditions the detection tests for

401 O2(1g) on the one hand and O2–, H2O2 and OH on the other, demonstrate the

402 simultaneous presence of all the mentioned ROS. The latter group of ROS is

403 generated after the electron transfer process (8).

404 4.3 Photoprotection of trp-photooxidation by diOHCh

405 A dominant O2(1g) physical scavenging ability by a given substrate represents a

406 positive feature in the context of oxidative interactions involving biomolecules. In

407 this case the considerably low values for the kr/kt quotient exhibited diOHCh and

408 diOHMeCh is a desirable quality. The final result is the elimination of O2(1g)

409 without appreciable loss of the scavenger.

410 This action constitutes a protection for proteins, DNA and other cell matrix

411 components.50 This photo-protective effect exerted by diOHCh was evident as

412 observed by the clear decrease in the rate of oxygen uptake for the system trp +

413 chalcone as compared to that of the amino acid alone, upon Rf-

414 photosensitization.

415 4.4 Final Remarks

416 The selective aerobic photoirradiation of Rf in the presence of ProAV produces

417 O2(1g) and promotes the generation of the ROS O2–, H2O2 and OH, as a

418 consequence of the respective energy transfer and electron transfer reactions

419 (16) and (8). All the ROS formed are quenched by ProAV in a dominant physical

420 fashion.
421 In quantitative terms ProAV shows an enhanced protective action as compared

422 with the well known synthetic ROS-scavenger trolox and constitutes an

423 interesting source for natural antioxidants extraction.

424

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