Riboflavin degradation in the presence of quercetin in methanol under continuous UV-B irradiation: The ESI-MS-UHPLC analysis

Article abstract of DOI:10.1007/s00706-015-1561-1

The presented work deals with continuous UV-B irradiation of riboflavin in MeOH solution, leading to its degradation under anaerobic as well as aerobic conditions (faster in the former case), which is related to riboflavin photosensitizing properties (type I photosensitizer in the first case, and type II in the other one). Addition of quercetin, a well-known antioxidant in the system causes a decrease of the (riboflavin) degradation in both cases. In anaerobic conditions it might be a consequence of quercetin antioxidant scavenging activity, while under aerobic conditions it could be related to singlet oxygen formation. The degradation dynamics - in both systems, in the presence and in the absence of quercetin - is well synchronized with dynamics formation of the two major products, lumiflavin and lumichrome

Full text of DOI:10.1007/s00706-015-1561-1

Monatsh Chem (2015) 146:1787–1794
DOI 10.1007/s00706-015-1561-1
ORIGINAL PAPER
Riboflavin degradation in the presence of quercetin in methanol
under continuous UV-B irradiation: the ESI–MS–UHPLC
analysis
1
Jelena S. Stanojevic Jelena B. Zvezdanovic Dejan Z. Markovic
1
1
•
•
´
´
´
Received: 12 May 2015 / Accepted: 17 August 2015 / Published online: 4 September 2015
Ó Springer-Verlag Wien 2015
Introduction
Abstract The presented work deals with continuous UV-
B irradiation of riboflavin in MeOH solution, leading to its
degradation under anaerobic as well as aerobic conditions
(faster in the former case), which is related to riboflavin
photosensitizing properties (type I photosensitizer in the
first case, and type II in the other one). Addition of quer-
cetin, a well-known antioxidant in the system causes a
decrease of the (riboflavin) degradation in both cases. In
anaerobic conditions it might be a consequence of quer-
cetin antioxidant scavenging activity, while under aerobic
conditions it could be related to singlet oxygen formation.
The degradation dynamics—in both systems, in the pres-
ence and in the absence of quercetin—is well synchronized
with dynamics formation of the two major products,
lumiflavin and lumichrome.
Solar energy reaching the Earth’s surface consists of pho-
tons from UV (100–400 nm), visible (400–800 nm), and
infrared (800 nm to *1 mm) range. UV light makes about
5 % in the total sunlight energy flux, it is conventionally
divided
into
UV-A
([315–400 nm),
UV-B
([280–315 nm), and UV-C ([100–280 nm) [1, 2]. The
most dangerous part of UV radiation is UV-B (representing
4–5 % of total UV flux reaching the Earth). Although it
stimulates vitamin D synthesis it may also induce a lot of
harmful effects including sunburns, inflammation,
immunosuppression, premature aging, and skin cancer [3].
Such detrimental effects are undoubtedly related to sun-
light absorption by certain molecules, called ‘‘endogenous
photosensitizers’’ located on various depths of skin layers.
These molecules, among others, include melanin and its
precursors, NADH/NADPH, tryptophan, trans-urocanic
acid, and flavins (riboflavin) [4, 5]. They are responsible
for the generation of light-driven Reactive Oxygen Species
(ROS), free radicals and singlet oxygen.
Graphical abstract
Riboflavin (vitamin B2; 7,8-dimethyl-10-ribityl-isoal-
loxazine) is one of the most important members of the huge
family of flavins—prosthetic group of flavoproteins—
characterized by the tricyclic heterocycle isoalloxazine
(Fig. 1). It belongs to a group of hydrosoluble vitamins
being an essential nutrient for humans. Because of humans’
inability to synthesize vitamin B2, it has been obtained
through diet. Riboflavin is predominantly found in milk,
eggs, meat products, cereals, fatty fish, dark-green veg-
etables, and fermented beverages [6]. It exists in three
forms: free riboflavin and two cofactor forms, flavin ade-
nine dinucleotide (FAD) and flavin mononucleotide
(FMN). It is used as a component of liquid preparations and
parenteral nutrition solutions [7]. Riboflavin (RFL) is
Keywords Riboflavin Á Photochemistry Á Pigments Á
Lumiflavin Á Lumichrome Á Mass spectrometry
´
& Jelena S. Stanojevic
jelena_stanojevic@yahoo.com
´
Jelena B. Zvezdanovic
jelite74@yahoo.com
1
ˇ
Faculty of Technology, University of Nis, Bulevar
Oslobodenja 124, 16000 Leskovac, Serbia
¯
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J. S. Stanojevic et al.
1788
Fig. 1 Major (a) and minor (b) products formed as a result of continual UV-B irradiation of riboflavin in methanol (according to the scheme
given by Insinska-Rak et al. [27])
known for its photosensitizing abilities in biological pro-
cesses, acting both as type I (free radical-) and type II
sensitizer [8]. One of the most important medical uses of
RFL photosensitizing property is in the novel therapy of
keratoconus called Corneal Collagen Crosslinking (CXL).
ROS generated during RFL photosensitization mediate
production of new covalent bonds between collagen
molecules, thus increasing biomechanical strength and
stiffness of the corneal stroma [9].
basal cells and dermis) and eyes (in the lens, with con-
centration of 4.5 lM) [11, 12], provoking damage to other
cell components. Riboflavin is capable of oxidizing a
variety of biomolecules including proteins (i.e., amino
acids, such as tryptophan) [13], carbohydrates [14], lipids
[15, 16], nucleic acids [17], as well as other vitamins [18,
19], through mixed type I and type II photosensitizing
mechanisms [20, 21]. In direct chemical quenching of its
singlet or triplet state (both states are reactive) [21] by
various substrates (such as phenolic compounds) RFL
donates or accepts hydrogen or electron, thus producing
free radicals (type I mechanism) which undergo further
reactions with oxygen or other organic molecules. Non-
radical type II mechanism includes physical quenching
(i.e., energy transfer) from the triplet-state RFL to
Due to its strong absorbance in the ultraviolet and vis-
ible region with absorbance maxima at 223, 267
(e = 32,500 M-1 cm-1), 373 (e = 10600 M-1 cm-1), and
444 nm (e = 12,500 M-1 cm-1) [10] its photoexcitation
may potentially occur in the organs and tissues most
exposed to light such as the skin (riboflavin is located in the
123

Riboflavin degradation in the presence of quercetin in methanol under continuous UV-B…
1789
molecular oxygen in its ground state (3O2) yielding very
Results and discussion
reactive singlet oxygen (1O2). (Pre)domination of one or
the other sensitizing mechanism depends upon the type of
substrate and reaction conditions such as pH, solvent,
concentration of oxygen, concentration of evolving com-
pounds, etc. [22]. Generally, low oxygen concentration
favors type I mechanism [23].
The effect of UV-B continuous irradiation on mixture of
riboflavin and quercetin in methanol solution is shown on
UHPLC chromatograms in Fig. 2a. The traces were
recorded at kdet = 385 nm. The top trace belongs to the
non-irradiated mixture, while the middle and the lower
trace belong to 6 min irradiated mixture under aerobic and
anaerobic conditions, respectively. The detection wave-
length of 385 nm was chosen based on absorption spectra
Due to the ability to accept and/or donate a pair of
hydrogen atoms and thus become reduced and/or oxidized
[10], riboflavin photochemistry is complex and can be
separated in three categories: intra- and intermolecular
photoreduction, intra- and intermolecular photoaddition,
and intramolecular photodealkylation. Some or all of the
photoreactions may occur simultaneously yielding cyclo
dehydroriboflavin (CDRF), formylmethylflavin (FMF),
lumichrome (LC), lumiflavin (LF), and many other
degradation products, depending on reaction conditions,
especially on solution pH value [24–26].
A recent study dealing with UV-A continuous irradia-
tion of riboflavin and its derivatives in aerobic and argon-
saturated methanol solution suggests that flavin triplet
states have been included in flavin degradation and prod-
ucts formation [27]; the authors postulated two different
mechanism, one including free radicals (in which flavins
react as type I sensitizer) and the other, non-radical type
involving singlet oxygen formation (permitted through the
flavins type II sensitization). They have also recorded LF-
and LC-type of compounds among the major products.
Overall they concluded that RFL photolysis was more
efficient under anaerobic conditions. This study presents a
step ahead since it deals with UV-B irradiation of ribo-
flavin in methanol solution in the presence of quercetin
(QC), well known for its antioxidant properties and
behavior; however, it can also act as a quencher of ribo-
flavin triplet state by electron transfer, and as a radical
scavenger [28, 29].
Both RFL and QC are present in epidermis skin layer
and both are excellent UV-B absorbers (e.g., endogenous
photosensitizers) [5], reducing therefore the amount of the
most dangerous sunlight fraction that hits the skin and
consequently the potential damage as well. However, the
governing mechanisms are still very little known; so the
picture of their potential interaction—mediated by UV-B—
is still pretty much obscure and barely more than a spec-
ulation. This work provides a basic data set for RFL-QC
interaction, in much simpler environment (MeOH solu-
tion), under continuous UV-B irradiation by using ultra
high-performance liquid chromatography–electrospray
ionization–mass spectrometry (UHPLC–ESI–MS) method,
and—the authors hope—could be of some relevance for the
analog picture in vivo.
Fig. 2 Riboflavin (RFL)–quercetin (QC) mixture in methanol, irra-
diated with continuous UV-B irradiation. The UHPLC
chromatograms for the non-irradiated and 6 min irradiated solution
(at aerobic and anaerobic conditions), recorded (wavelength detec-
tion) at 385 nm (a). Part of the chromatogram recorded at 300 nm:
10 min UV-B irradiated RFL-QC mixture under aerobic conditions
(b). Absorption spectra of riboflavin (peak No. 1, tret = 2.41 min,
from a) and UV-B-induced products, 3 and 7, e.g., lumiflavin (LF,
peak No. 3, tret = 3.26 min) and lumichrome (LC, peak No. 7,
tret = 6.56 min) (c) from the chromatogram shown in the (a)
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J. S. Stanojevic et al.
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Fig. 3 The MS/MS spectra of riboflavin—RFL (peak No. 1 from UHPLC chromatograms shown in Fig. 2a) (a) and the products, lumiflavin
(LF) and lumichrome (LC), the peaks No. 3 and 7 (b) and (c), respectively
of RFL (peak No. 1, tret = 2.41 min), LF (peak No. 3,
tret = 3.26 min) and LC (peak No. 7, tret = 6.56 min),
shown in Fig. 2c; evidently the all three species have a
strong absorption at 385 nm. The peaks No. 2, 4, and 6
(from Fig. 2a) have not been identified, though the latter
two have a LF-type of structure (based on the absorption
spectra). The peak No. 5 belongs to formyl methyl flavin,
FMF (Fig. 1b). Part of the chromatogram from
tret = 4–8 min was recorded at kdet = 300 nm (Fig. 2b).
The intention was to record possible QC degradation
products found also in the other UV-irradiation studies
done in MeOH solution—with absorption maximum at
300 nm [30–32]. Evidently, the two new peaks appeared,
No. 9 and 10; based on the absorption spectra, peak 10 is
very similar to the one proposed in the earlier work [31],
resulting from the opening from the QC B-ring.
irradiation wavelengths on RFL photodegradation reactions
(photoaddition and photoreduction) in solution and in the
presence of phosphate buffer, using UV and visible light
sources. They have also found LC and LF among the major
products and reported that the ratio LF/LC increased with
the change from UV to VIS radiation. That is in good
correspondence with the data presented in this report: it is
clear that LC dominates over LF after 6 min of UV-B
irradiation under anaerobic conditions (LF is not even
recorded after the analog irradiation under aerobic condi-
tions; Fig. 2a).
Time dynamics of riboflavin degradation in MeOH
during UV-B irradiation regime, in the presence and in the
absence of quercetin, under anaerobic and aerobic condi-
tions, is shown in Fig. 4a; to be able to better understand
quercetin influence, QC degradation was traced alone as
well in the mixture with RFL—both under anaerobic and
aerobic conditions (Fig. 4b).
The MS/MS spectra (obtained by using tandem mass
spectrometry with collision induced dissociation of the
molecular ions) shown in Fig. 3 confirm the structure of the
two major products, lumiflavin (Fig. 3b—related to the
peak No. 3 on chromatogram in Fig. 2a), and lumichrome
(Fig. 3c—the peak No. 7 in Fig. 2a). Riboflavin itself
yields LC as the major fragmentation product (Fig. 3a); the
following fragmentation involves cleavage of the isoal-
loxazine entity third ring—through a release of the
CONHCO neutral fragment, and that is the same pattern
seen in the MS/MS spectra of LC itself (Fig. 3c). On the
other hand, according to LF MS/MS spectra the CONHCO
elimination occurs in two consecutive steps yielding frag-
ments at m/z = 214 ([MH-CONH]?) and m/z = 186 (the
additional loss of neutral CO fragment) (Table 1). Ahmad
and coworkers [33] studied the effect of light intensity and
During first 15 min of UV-B irradiation QC appears
to be much more resistant than RFL in aerobic condi-
tions; RFL itself degrades much faster in anaerobic
conditions (Fig. 4a, b). It is clear that QC acts as a RFL
protective factor toward the applied UV-B irradiation,
e.g., the order in dynamics of RFL degradation looks as
follows: RFL-anaer. [ (RFL?QC)-anaer. [ RFL–aer. [
(RFL?QC)-aer. (Figure 4a). Synchronously, dynamics
ordering in QC decrease with increasing irradiation
periods is: QC \ (RFL?QC)-anaer. \ (RFL?QC)-aer.
(Fig. 4b).
The different RFL degradation under anaerobic and
aerobic conditions (in the absence of QC) is clearly dic-
tated by RFL photosensitizing properties [20, 21]. The
123

Riboflavin degradation in the presence of quercetin in methanol under continuous UV-B…
1791
Table 1 The MS/MS spectra obtained by ESI–MS method for riboflavin, quercetin, and some degradation products obtained as a result of UV-B
irradiation of (RFL?QC) mixture in MeOH solution
b
Peak
tret/min
k
max/nm
m/z [M - H]-
m/z [M ? H]?
MS/MS of [M ? H]?/c[M - H]-
1
2
1
2
3
4
5
6
9a
7
2.41
2.58
3.26
3.64
3.90
4.81
5.11
6.56
6.49
371
446
370
445
365
445
368
445
375
449
373
450
295
375
–
377
359b, 243 (100 %), 216, 198, 172
6.76
7.60
8.01
8.30
8.93
375
243b
255
–
257
214b, 186 (100 %)
283
-
283
–
285
253b
-
363
241
365
347b, 333 (100 %)
331c
216b, 198, 172
9.99
353
385
300
260
372
243 (100 %)
10a
8
7.20
7.60
347
301
349
303
211c
285b
a
b
Products of quercetin; MS/MS-fragment ions in positive and negative mode
c
fastest RFL degradation in the absence of quercetin and
under anaerobic conditions is clearly related to its type I
photosensitizing activity that takes place via its triplet state
and involves formation of semiquinone RFL-radical [27].
In the presence of QC—and also under anaerobic condi-
tions—RFL degrades a bit slower. It is well known that QC
expresses its antioxidant ability toward UV-induced effects
in two different ways, (1) through a strong UV absorption
[34, 35], and/or (2) chain-breaking activities, i.e., by
scavenging present radicals, for example by preventing
oxidative skin damage [36–38]. Though one may reason-
ably speculate that the both ways could contribute to
decrease of RFL degradation (both RFL and QC are
excellent UV-B absorbers) [5], the latter one—through
possible scavenging of UV-created RFL radicals—would
certainly affect rate of RFL degradation, e.g., decrease it,
which is shown in Fig. 4a. In the same time a clear
decrease of QC concentration was seen under anaerobic
conditions in the presence of RFL, compared to the RFL-
absence case (Fig. 4b).
(Fig. 4a). Clearly, QC contributes to decrease of RFL
degradation both under anaerobic and aerobic conditions,
but more in the latter case (Fig. 4a, b). The same conclu-
sion is drawn by tracing change of QC concentration in the
presence of RFL, under anaerobic and aerobic conditions
(including change of QC concentration under aerobic
conditions in the absence of RFL; Fig. 4b). There, RFL
certainly acts as the type II photosensitizer, so the protec-
tion must include involvement of singlet oxygen formation
[27].
The plots tracing dynamics of appearance of the two
major products, lumiflavin and lumichrome (Fig. 5a, b) are
generally in coherence with the above presented data,
though it looks a bit more consistent with lumiflavin
(Fig. 5a). Lumiflavin structure is more similar to RFL than
lumichrome (Fig. 1), though in both cases the isoalloxazine
entity has been survived the applied UV-B irradiation
regime. The RFL anaerobic degradation in 8 min (irradi-
ation) period (Fig. 4a, the lowest trace) is matched by a
synchronous exponential-shape type of LF rise in the same
(anaerobic) conditions (Fig. 5a, the upper trace). The same
type of rise but with earlier achieved plateau has been seen
when QC was added in the system (Fig. 5a, the middle
trace)—and this well matches with the RFL anaerobic
degradation in the presence of QC (see Fig. 4a). Finally, in
the aerobic conditions LF expresses the slowest, almost
The protective effect of QC (toward RFL) is also seen
under aerobic conditions. First of all, it is evident that RFL
degradation itself under aerobic conditions is slower
compared to the anaerobic conditions (in the absence and
in the presence of QC, as well). The addition of QC in the
aerobic system makes RFL degradation even more slower
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J. S. Stanojevic et al.
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Fig. 4 Degradation of riboflavin and quercetin in both aerobic and
anaerobic conditions under continuos UV-B irradiation regime
(particularly, and in the mixture), expressed as the changes in
percents of RFL and QC (compared to non-irradiation) for different
irradiation time periods (tirr)—e.g., the dynamic plots (a and b,
respectively). The plots are based on the data from DAD-recorded
chromatograms (the peaks No. 1 and 8, at tret = 2.41 and 7.80 min,
respectively, shown in the Fig. 2a), at 446 and 372 nm, e.g.,
absorption maximums for RFL and QC, respectively. The percents
of RFL and Querc. were calculated by using equation: Content
(%) = (At 9 100)/A0, where A0 and At representing the peak area
values of RFL and QC at the corresponding tret values, for tirr = 0,
and different irradiation periods, respectively
Fig. 5 The changes in peak areas for LF and LC (the peaks No. 3 and
7 at tret = 3.26 and 6.56 min, respectively, from chromatogram shown
in Fig. 2a) for different irradiation time periods (tirr)—e.g., dynamic
plots (a and b, respectively). The plots are based on the data from
DAD-recorded chromatograms at 445 and 385 nm, e.g., absorption
maximums for LF and LC, respectively
(on a bit longer irradiation scale) the undoubtedly proven
QC preventive effect on RFL degradation (Fig. 4a, b) is
once again confirmed: more LC is formed in the absence
than in the presence of QC (Fig. 5b). Still, a question
emerges: why in the same (RFL?QC)-aerobic system, and
under the same UV-B irradiation regime, one of the two
major products (LC) is being formed, while the other one
(LF) is absent (Fig. 2a, the middle and the lowest traces)?
The question is quite relevant having in mind that the both
products have very similar structures, preserving basic
isoalloxazine entity following the irradiation (Fig. 1); it is
hard to see how the minor structural differences, presence
(LF) or absence (LC) of internal conjugation inside it—
though leading to different spectral behavior (Fig. 2c)—
may such differently favor formation of the two products.
Still, what looks undoubtful is that this behavior must
linear type of rise (Fig. 5a, the lowest trace), which is
nicely matched with RFL aerobic degradation pattern
(Fig. 4a). Only one thing makes the exception from this
synchronicity between RFL degradation and LF produc-
tion: when QC is added to RFL under aerobic conditions
there is no any trace of LF presence (see Fig. 2a, the
middle trace).
Lumichrome dynamics plots (Fig. 5b) expressed very
similar behavior to lumiflavin (Fig. 5a). The same pattern
has been seen under anaerobic conditions during the same
8-min irradiation period, while in the aerobic conditions
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Riboflavin degradation in the presence of quercetin in methanol under continuous UV-B…
1793
Ultra high-performance liquid chromatography-
diode array-electrospray ionization mass
spectrometry analysis
include QC action, and most probably involvement of
singlet oxygen, certainly created in the system by RFL type
II photosensitizing activity [27]: the plots in Fig. 4b sup-
port this explanation.
The chromatography runs were carried out using a Dionex
Ultimate 3000 UHPLC? system equipped with a diode
array (DAD) detector and connected to LCQ Fleet Ion Trap
Mass Spectrometer, Thermo Fisher Scientific, USA. The
separations were performed on a Hypersil gold aQ C18
column (150 9 3 mm, 3 lm) at 25 °C.
To conclude, this work confirms that continuous UV-B
irradiation of riboflavin in MeOH solution leads to its
degradation both under anaerobic and aerobic conditions
(faster in the former case) which is related to RFL sensi-
tizing properties (type I photosensitizer in the first case,
and type II in the other one). Addition of quercetin in the
system leads to decrease of the (RFL) degradation in both
cases; in anaerobic conditions it certainly might include
QC antioxidant scavenging activity while under aerobic
conditions the degradation decrease should be related to the
involvement of singlet oxygen formation. The degradation
dynamics—in both systems, in the presence and in the
absence of QC—is well synchronized with dynamics for-
mation of the two major products, lumiflavin and
lumichrome. The unappearance of LF in the aerated
(RFL?QC) system still needs to be explained.
The mobile phase consisted of (A) 0.1 % formic acid in
water and (B) 0.1 % formic acid in methanol. The next
linear gradient program at flow rate of 0.5 cm3/min has
been applied to RFL in methanol only: 20–50 % (B) for the
first 6.5 min, followed by isocratic 50 % (B) from 6.5 to
10th min, 50–20 % (B) from 10 to 10.1 min, and finally
isocratic run with 20 % (B) to 20th min. Regarding RFL-
QC and QC solutions in methanol the next linear gradient
program has been applied: 40 % (B) for the first 2.5 min,
40–95 % (B) from 2.5 to 5.5 min, isocratic run with 95 %
(B) for the next 1.5 min, 95 to 40 % (B) from 7 to 7.1 min,
and finally isocratic run with 40 % (B) to 13th min. The
injection volume was 0.5 mm3. Absorption UV–vis spectra
were recorded on DAD-detector (200–800 nm), set at four
detection wavelengths, kdet. (for RFL solution): 445 nm
(RFL absorption maximum, Amax), 410 nm (for CDRF
detection), 385 nm (for FMF detection) and 356 nm (for
LC detection). For RFL-QC solution kdet. was set at:
445 nm, 410 nm, 385 nm (for the same reasons as in the
former case) and 372 nm (QC absorption maximum, Amax);
finally, for QC sample only: 255 nm (QC Amax—band II),
295 nm and 300 nm (QC degradation products Amax) and
372 (QC Amax—band I), simultaneously. The MS-analysis
was performed using a LCQ 3D-ion trap mass spectrometer
with electrospray ionization (ESI) in both positive and
negative ion mode. For positive ion mode, the ESI-source
parameters were set as follows: source voltage 4.5 kV,
capillary voltage 22 V, tube lens voltage 65 V, capillary
temperature 350 °C, sheath and auxiliary gas flow (N2) 60
and 5 (arbitrary units), respectively. For negative ion mode,
source voltage was set to 4.5 kV, capillary voltage -60 V,
tube lens voltage -95 V, capillary temperature 350 °C,
sheath and auxiliary gas flow 42 and 7, respectively. MS
spectra were obtained by full range acquisition of m/
z = 150–1000 and 100–650. For fragmentation study (MS/
MS), a data dependent scan was performed by deploying
the collision–induced dissociation (CID). The normalized
collision energy of the CID cell was set at 15 and 17 eV.
Experimental
Sample preparation
Riboflavin and quercetin stock solutions were prepared by
dissolving 36.7 mg RFL (98 %, Alfa Aesar, Karlsruhe,
Germany) in 25 cm3 of 0.01 M NaOH (AnalaR NOR-
MAPUR, Leuven, Belgium) in redestilated water, and
3.02 mg of quercetin in 10 cm3 of methanol (J.T. Baker,
BAKER ANALYZED, LC–MS reagent, Deventer, The
Netherlands). The sample solutions were made by diluting
the stock solution with methanol; they are stored at 4 °C
until their use, protected from light with aluminum foil (to
avoid any photochemical changes). The concentration of
riboflavin as well as quercetin were 0.1 mM in the final
methanol solutions with pH = 8.69. All experiments were
done at room temperature.
UV-irradiation treatment
Continuous UV-irradiations of the riboflavin samples (un-
der aerobic and anaerobic conditions) in methanol were
performed in cylindrical photochemical reactor ‘‘Rayonet’’,
with 10 symmetrically placed lamps with emission maxi-
mum at 300 nm (UV-B). The samples were irradiated in
quartz closed cuvettes (1 9 1 9 4.5 cm) placed on rotat-
ing circular holder. The total measured energy flux was
15.0 W/m2 at 10 cm distance. The anaerobic conditions
were achieved by bubbling the solutions with nitrogen for
at least 10 min prior to the irradiation.
Acknowledgments This work was supported under Projects of the
Ministry of Education, Science and Technological development of the
Republic of Serbia, Projects No.TR-34012 and OI-172044.
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19. King JM, Min DB (2002) J Am Oil Chem Soc 79:983
20. Sikorska E, Khmelinskii I, Komasa A, Koput J, Ferreira LFV,
References
´
Herance JR, Bourdelande JL, Williams SL, Worrall DR, Insin-
1. Young A (2006) Prog Biophys Mol Biol 92:80
2. International Agency for Research on Cancer (2006) Exposure to
artificial UV radiation and skin cancer. IARC working group
reports No 1. IARC
3. Schade N, Esser C, Krutmann J (2005) Photochem Photobiol Sci
3:699
4. Longstreth J, de Gruijl FR, Kripke ML, Abseck S, Arnold F,
Slaper HI, Velders G, Takizawa Y, van der Leun JC (1998) J
Photochem Photobiol B Biol 46:20
5. Wondrak GT, Jacobson MK, Jacobson EL (2006) Photochem
Photobiol Sci 5:215
6. Powers HJ (2003) Am J Clin Nutr 77:1352
´
7. Silva E, Gonzalez T, Edwards AM, Zuloaga F (1998) Nutr
Biochem 9:149
8. Girotti AW (2001) J Photochem Photobiol 63:103
9. Makdoumi K (2011) Ultraviolet light A (UVA) photoactivation
of riboflavin as a potential therapy for infectious keratitis. Ph.D.
ska-Rak M, Sikorski M (2005) Chem Phys 314:239
21. Cardoso DR, Libardia SH, Skibsted LH (2012) Food Funct 3:487
22. Heelis F (1982) Chem Soc Rev 11:15
23. Min DB, Boff JM (2002) Compr Rev Food Sci Food Saf 1:58
24. Ahmad I, Fasihullah Q, Vaid FHM (2004) J Photochem Photobiol
B Biol 75:13
25. Ahmad I, Fasihullah Q, Vaid FHM (2005) J Photochem Photobiol
B Biol 78:229
26. Ahmad I, Ahmed S, Sheraz MA, Vaid FHM, Ansari IA (2010) Int
J Pharm 390:174
27. Insinska-Rak M, Golczak A, Sikorski M (2012) J Phys Chem A
116:1199
28. Huvaere K, Olsen K, Skibsted LH (2009) J Org Chem 74:7283
29. Becker EM, Cardoso DR, Skibsted LH (2005) Eur Food Res
Technol 221:382
30. Fahlman BM, Krol ES (2009) J Photochem Photobiol B Biol
97:123
´ ´
31. Zvezdanovic JB, Stanojevic JS, Markovic DZ, Cvetkovic DJ
¨
¨
Thesis, Orebro University, Orebro
´
´
10. Choe E, Huang R, Min DB (2005) J Food Sci 70:R28
11. Kino K, Kobayashi T, Arima E, Komori R, Kobayashi T,
Miyazawa H (2009) Bioorg Med Chem Lett 19:2070
12. de La Rochette A, Silva E, Birlouez-Aragon I, Mancini M,
(2012) J Serb Chem Soc 77:297
´
´
(2012) J Serb Chem Soc 77:1571
´ ´
32. Zvezdanovic JB, Markovic DZ, Cvetkovic DJ, Stanojevic JS
33. Ahmad I, Fasihullah Q, Vaid FHM (2006) J Photochem Photobiol
B Biol 82:21
34. Strid A, Porra RJ (1992) Plant Cell Phys 33:1015
35. Strid A, Chow WS, Anderson JM (1994) Photosynth Res 39:475
36. Schoemaker JH, Schoemaker MT, Zijlstra H, van der Horst FA
(1995) Dermatology 191:36
37. Casagrande R, Georgetti SR, Verri WA Jr, Dorta DJ, dos Santos
AC, Fonseca MJV (2006) J Photochem Photobiol B: Biol 84:21
38. Choquenet B, Couteau C, Paparis E, Coiffard LJM (2008) J Nat
Prod 71:1117
`
Edwards A-M, Morliere P (2000) Photochem Photobiol 72:815
13. Silva E, Ugarte R, Andrade A, Edwards AM (1994) J Photochem
Photobiol B Biol 23:43
14. Silva E, Edwards AM, Pacheco D (1999) J Nutr Biochem 10:181
15. Chien JT, Lu YF, Hu PC, Chen BH (2003) Food Chem 81:421
16. Huvaere K, Cardoso DR, Homem-de-Mello P, Westermann S,
Skibsted LH (2010) J Phys Chem B 114:5583
17. Joshi PC, Keane TC (2010) Biochem Biophys Res Commun
400:729
18. Akhtar MJ, Khan MA, Ahmad I (2000) J Pharm Biomed Anal
23:1039
123