Contents lists available at

Journal of Photochemistry and Photobiology B: Biology

journal homepage:

Investigations of riboflavin photolysis via coloured light in the nitro blue

tetrazolium assay for superoxide dismutase activity
⇑
Chien-wei Cheng, Liang-yü Chen, Chan-wei Chou, Ji-yuan Liang
Department of Biotechnology, Ming-Chuan University, Gui-Shan 33343, Taiwan

articleinfo
abstract
Article history:
Received 27 January 2015 Determination of the superoxide dismutase activity is an important issue in the fields of biochemistry
Accepted 30 April 2015 and the medical sciences. In the riboflavin/nitro blue tetrazolium (B2/NBT) method, the light sources
Available online 8 May 2015 used for generating superoxide anion radicals from light-excited riboflavin are normally fluorescent
lamps. However, the conditions of B2/NBT experiments vary. This study investigated the effect of the
Keywords: light source on the light-excitation of riboflavin. The effectiveness of the photolysis was controlled by
Superoxide dismutase the wavelength of the light source. The spectra of fluorescent lamps are composed of multiple colour
LED lights, and the emis- sion spectra of fluorescent lamps made by different manufacturers may vary. Blue
Blue light light was determined to be the most efficient for the photochemical reaction of riboflavin in visible
NBT region. The quality of the blue light in fluorescent lamps is critical to the photo-decomposition of
Riboflavin
riboflavin. A blue light is better than a fluorescent lamp for the photo-decomposition of riboflavin. The
performance of the B2/NBT method is thereby optimized.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction
laboratories [6]. Beyer and Fridovich [7] investigated the effects
of experimental variables on two indirect assays, the xanthine oxi-
Superoxide dismutase (SOD) is essential to many living organ-
dase and cytochrome C method and the riboflavin/nitro blue tetra-
isms. This enzyme is one of the most important antioxidant
zolium method (B2/NBT). The B2/NBT method is considered
defence mechanisms in microorganisms and plant cells that are
simpler and is preferred for the quantification of SOD activity in
exposed to oxygen [1]. The antioxidant capacity of natural ingredi-
crude extracts [7].
ents is also a significant issue for foods with special dietary pur-
Riboflavin, also known as vitamin B 2, is very sensitive to light. It
poses [2]. SOD, as a free radical scavenger, can convert
decomposes after being irradiated by ultraviolet (UV) or visible
superoxide anion radicals (OH—) into H O and O in living cells. light (420–560 nm) for a very short time [8], generating free radi-
2 2 2 2
By scavenging OH—, the oxidation of lipid membranes can be
2

pre- vented [3]. Superoxide anion radicals are intermediate ⇑ Corresponding author. Tel.: +886 3 3507001x3772; fax: +886 3 3593878.
products generated during oxidation or reduction. Formed from E-mail address: jiyuanl@gmail.com (J.-y. Liang).
hydroxyl radicals or hydroxyl peroxide compounds,
2 OH— causes
damage, inflammation, atherosclerosis and aging of cells [4,5].
Hence, the determination of SOD activities, either in vivo or in
vitro, is an important topic in the fields of biochemistry and
the medical sciences.
Many methods, both direct and indirect, have been
developed for the determination of SOD activity. However,
direct assays for SOD determination are scarce because of their
need for special apparatus, such as an electron paramagnetic
resonance spectrom- eter (EPR). Indirect assays relying on the
ability of SOD to inhibit OH—-driven reactions are more widely
2
applied in biochemical
cals of reactive oxygen species (ROS), such as OH— and singlet
oxy- gen [9]. The riboflavin photochemical treatment with blue
light can be employed to inactivate E. coli with generated ROS
[10,11].
Superoxide anion radicals generated from light-excited
ribofla- vin can be utilized to examine the effect of luminance
on light reac- tions of nitro blue tetrazolium (NBT) [5]. In this
study, NBT is used as an indicating scavenger to be reduced
by OH—. NBT reduction causes an increase in the absorbance
at 560 nm in a process that may be inhibited by SOD. In
http://dx.doi.org/10.1016/j.jphotobiol.2015.04.028
1011-1344/© 2015 Elsevier B.V. All rights reserved.
addition, the B2/NBT method can be employed to evaluate the
contents of phenolic compounds in func- tional foods through ROS
2
scavenging.
In the B2/NBT method, the light sources used for generating OH—
2
from light-excited riboflavin are normally fluorescent lamps. A flu-
orescent lamp is excited to generate ultraviolet rays by
a low-pressure mercury vapour and argon. The inner wall of a
glass tube is coated with a fluorescent substance and
2
stimulated by ultraviolet rays to generate a hybridized visible
light. However,
 C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
the conditions and apparatuses for the B2/NBT experiments varied
in the studies reviewed. The fluorescent lamps used were of differ-
ent specifications, including 13 W [1], 15 W [12,13], 20 W [7,14],
25 W [15], 30 W [16] and 40 W [17], and were illuminated over
different distances and for different durations. The reactions were
initiated by adding riboflavin at 2000 [18], 3000 [19], 4000 [20]
and 5000 lux [21] for the luminance of fluorescent lamps.
The effects of light source properties, such as colour and wave-
length, on the light-excitation of riboflavin have been investigated
using the B2/NBT assay [22]. The fluorescent lamp is excited to
gen- erate a hybridized visible light comprised of multiple colour
lights. Thus, the properties of the light source could affect the
riboflavin photochemistry, leading to incorrect conclusions from
the B2/NBT assay. To ensure high accuracy, the widely accepted
B2/NBT assay for the quantification of SOD activity has to be
validated.
The current study developed an effective SOD assay from the
B2/NBT method by applying a well-defined light source to ribofla-
vin photochemical reactions. The goal was to investigate the effects
of light quality on the light-excitation of riboflavin as assayed by
the B2/NBT method. The results thus obtained would promote
the consistency of enzymatic measurements using photolysis
reactions.
2. Materials and methods
2.1. Setup of illumination units
The photo-induced reactions were performed in a plastic box
(104 cm 74 cm 55 cm) with a light source. The box was made
× ×
of white cardboard, and its outer surface was covered with black
cloth. Three light-emitting diode (LED) tube lights (580 mm
length) in red, green and blue (VITALUX T8HO LED tube lights,
Vita LED Technologies Co., Tainan, Taiwan) and two fluorescent
lamps, Fluor-A (38 W, FHF38WEX, Taiwan Fluorescent Lamp Co.,
Taipei, Taiwan) and Fluor-B (30 W, FCL30D/28, China Electric
MGF. Co., Taipei, Taiwan), were used as light sources. Irradiance
was measured by the power of the electromagnetic radiation per
unit area (mW/cm2) with radiometry and validated by a solar
power meter (TM-207, Tenmars Electronics Co., Taipei, Taiwan).
Luminance, a term used in photometry, was measured in lux (lx)
or lm/m2 by a digital light meter (YF-170, Tenmars Electronics
Co., Taipei, Taiwan).
2.2. Chemicals
Gallic acid, l-methionine, monopotassium phosphate, potas-
sium dihydrogen phosphate, riboflavin and SOD (S9697-15KU)
were purchased from Sigma–Aldrich (St. Louis, MO). The SOD
was assayed by Sigma–Aldrich using the xanthine oxidase/cy-
tochrome C method [23]. Nitro blue tetrazolium (NBT) was pur-
chased from Bio Basic, Inc. (Markham, Ontario, Canada). Ultra-
pure deionized water from a Milli-Q system was used as a solvent
in this study.
2.3. Spectrometry of light sources and riboflavin
The emission spectra of the fluorescent lamps and LED tube
lights were measured using a UV–vis miniature fibre optic spec-
trometer (USB4000 UV/Vis, Ocean Optics, USA) and were normal-
ized, as shown in Fig. 1. The wavelengths of the emitted maxima
of the blue, green, yellow and red lights were 463, 529, 589 and
632 nm, respectively, and the spectral widths at half height
(W1/2) were 23, 31, 16 and 14 nm, respectively. The spectra of
the fluorescent lamps are usually comprised of several peaks, as
shown in Fig. 1.
263
Fig. 1. Visible spectra of fluorescent and LED lamps used in this study.
The riboflavin (2.4 lM in 50 mM, pH 7.8 phosphate buffer) was
irradiated by the fluorescent lamps and LED tube lights at
1.0 mW/cm2 for 20 min. The absorbance of the illuminated ribofla-
vin was detected at 200–800 nm by a UV/vis spectrometer
(Lambda35, Perkin-Elmer).
2.4. Effects of light sources on the generation of O H— with the B /NBT
2
method
The reduction in NBT was determined using the method devel-
oped by Beauchamp and Fridovich [24]. All solutions were 50 mM
in phosphate buffer (pH 7.8). 3 mL of reactant was used, and the
concentrations of riboflavin, methionine and NBT were
2.4 10—6 M, 0.01 M and 1.6 10—4 M, respectively. The
× ×
distance between the reactant and the lamps was fixed, and the
irradiance was controlled. The reactant was illuminated by blue,
green, yel- low or red LED irradiation at 1.0 mW/cm2, by the
fluorescent lamps at 1.0 mW/cm2, and by the blue light irradiation
at 0.1 mW/cm2 for 10, 20 or 30 min. For the control treatment, the
reactant was kept in the dark. The photo-chemically reduction of
riboflavin generated OH—, which reduced NBT to form blue
2
formazan, which can be detected at 560 nm (Lambda35, Perkin-
Elmer).
2.5. Effects of light source on OH— scavenging activity using gallic acid
2
Gallic acid was employed to determine the effects of the
light source on the OH— scavenging activity using the B /NBT
2
method described in Section 2.4. In brief, gallic acid (50 lL) was
added to
3 mL reactant to final concentrations of 0, 10, 20, 40, 60, 80 and
100 lg/mL. Then, the mixed solutions were subjected to Fluor-A
or Fluor-B irradiation at 1.0 mW/cm2 or blue-light irradiation at
0.1 mW/cm2 for 20 min. Gallic acid can inhibit NBT
reduction, and the scavenging capacity of the OH— generated was
2
calculated using the following equation, where A denotes the
absorbance of the blue formazan measured at 560 nm.
Σ control — Asample Þ=Acontrol × 100%
OH— scavenging activityð%Þ ¼ ðA
2
ð1Þ
2.6. Effects of blue light on SOD activity
The effects of blue light on OH— scavenging activity were exam-
2
ined with the B2/NBT method using SOD, as described in Section
2.4. In brief, (A) 50 lL SOD was added to 3 mL reactant, and the
final activity of SOD (1.0 unit/g) was used as a standard.
Then, the mixed solutions were subjected to blue light irradiation
264 C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267

at 0.08, 0.1 or 0.12 mW/cm 2 for 10, 20 or 30 min. SOD activity was
defined as one unit of SOD having a 50% inhibition on the B 2/NBT
system. (B) 50 lL SOD was added to 3 mL reactant, and the
final
activity of SOD was 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0 or 4.0 unit/g.
The mixed solutions were exposed to blue light irradiation at
0.12 mW/cm2 for 10, 20 or 30 min. The optimal condition for
the 50% inhibition of OH— scavenging activity with SOD (1.0
2
unit/g) was measured using the B2/NBT method.

2.7. Statistics

Data are represented by the mean ± standard deviation of three

separate experiments. A homoscedastic two-sample t-test was
employed to assess whether the two sets of measurements dif-
fered, and values of P < 0.05 were considered to be significant.

3. Results

3.1. Effect of light quality on photo-decomposition of riboflavin

The generation of OH— from the intermediates during the decom-

2
position of riboflavin in aqueous solution was detected using
NBT reduction [7]. As shown in Fig. 2, the absorbance of the NBT
reduc- tion increased with the generation of OH— by the
2
photochemical system in the presence of riboflavin. For the
photochemical treat- ment, the decomposition of riboflavin
increased with the irradia- tion time. The highest efficiency
photochemical reaction of riboflavin was observed under blue
light irradiation. The average photochemical effects of the green,
yellow and red lights relative to the blue light were
approximately 4.9%. 3.9% and 2.6%, respec- tively. These results
indicate that the effectiveness of riboflavin photolysis is mainly
determined by the wavelength of light used and that the light
quality is associated with the photochemical reaction of
riboflavin.
3.2. Spectra of riboflavin in photoreactions
Fig. 3 shows the spectra (200–800 nm) of riboflavin measured
by fluorescence lamps and blue light irradiation. As observed,
there were four absorption peaks, 224, 268, 373 and 445 nm, of
ribofla- vin in the dark. The absorbance of riboflavin at 445 nm
was dra- matically decreased by blue light irradiation. The
fluorescent
Fig. 3. Absorption spectra of riboflavin irradiated by different light sources at
1.0 mW/cm2 for 20 min.
lamps had a very small impact because the variations in the spec-
tra at 445 nm were not significant. Blue light irradiation for 20 min
yielded the highest efficiency in the photo-decomposition of
riboflavin.
The spectra of riboflavin were measured during the course of
colour illuminations in the photo-decomposition reactions [11].
Irradiation with blue light showed the highest efficiency photo-
decomposition of riboflavin, while the absorbance of ribofla- vin at
445 nm decreasing dramatically upon illumination. The green,
yellow and red lights had negligible effects because the spectral
changes were not significant.
Fig. 4 shows the effects of the irradiation of fluorescent lamps
and blue light on the NBT reduction during the photochemical
reaction of riboflavin. As observed, the photochemical reaction of
riboflavin increased with the irradiation time. The

Fig. 2. Effects of colour-light irradiation at 1.0 mW/cm 2 on NBT reduction for 10, 20
and 30 min. Data are represented by mean ± standard deviation, where n = 3.
Significant differences (p < 0.05) between groups are indicated by different letters
above the bar. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
Fig. 4. Effects of fluorescent lamp irradiation at 1.0 mW/cm 2 and blue light
irradiation at 0.1 mW/cm 2 on NBT reduction for 10, 20 and 30 min. Data are
represented by mean ± standard deviation, where n = 3. Significant differences
(p < 0.05) between groups are indicated by different letters over the bar. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
 C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
photo-decomposition of riboflavin was lower under Fluor-A than
under Fluor-B irradiation at 1.0 mW/cm2.
3.3. Effect of light source on O H—
scavenging activity using gallic acid
2
Gallic acid is a tri-hydroxyl-benzoic acid. Phenolic
compounds are considered the most important antioxidants
in plants and plant-based foods [25]. Many phenolic
compounds, such as gallic acid, can remove free radicals
through a process similar to SOD-catalysed reactions. The
free radicals serve as a substrate and are scavenged by the
phenolic compounds. Fig. 5 shows the variation in OH— scavenging
2
activity (%) using different levels of gal- lic acid. As seen in Fig.
5(A), the O scavenging activity 2increased with the addition of
H—
gallic acid and was higher under Fluor-A irra- diation than under
Fluor-B.
A characteristic concentration of chemicals, IC 50, could be deter-
mined to provide 50% inhibition activity from the correlation
curve of the scavenging activity to the concentration. The IC 50 of
gallic acid is defined as the equivalent concentration of gallic acid
that is able to remove 50% of the superoxide anion radicals. As
shown in Fig. 5(B), the IC50 of gallic acid under Fluor-A and Fluor-B
irradi-
ation at 1.0 mW/cm2 was 25.7 lg/mL and 53.1 lg/mL, respectively,
while the IC50 of gallic acid under blue-light irradiation at
0.1 mW/cm2 was 45.1 lg/mL. The IC50 of gallic acid
under blue-light irradiation at 0.1 mW/cm 2 and Fluor-B
irradiation at
1.0 mW/cm2 were both insignificant.
Fig. 5. (A) OH— scavenging activity of gallic acid under fluorescent lamp
2
irradiation at 1.0 mW/cm2 and blue light irradiation at 0.1 mW/cm2 for 20 min. (B)
The IC50 of gallic acid under fluorescent lamp irradiation at 1.0 mW/cm2 and
blue light irradiation at 0.1 mW/cm2 for 20 min. Data are represented by mean
± standard deviation, where n = 3. Significant differences (p < 0.05) between
groups are indicated by different letters over the bar. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version
of this article.)
265
3.4. Effect of blue light on SOD detection
The OH— scavenging activity of one unit of SOD was determined
2
by blue light irradiation using the B2/NBT method. As shown in
Fig. 6(A), the OH— scavenging activity of one unit of SOD decreased
2
with an increase in the blue-light irradiation time for the
photo- chemical treatment.
The effect of the SOD activity on OH— scavenging activity
2
under blue-light irradiation were investigated using the B2/NBT
method. As shown in Fig. 6(B), the OH— scavenging activity
2
increased with the SOD activity, and the 50% inhibition value of the
2
O scavenging activity at 0.12 mW/cm2 blue-light irradiation
H—
for 10, 20 and 30 min were 0.82, 0.90 and 0.97 unit SOD,
respectively. The SOD activity is defined as such that one unit of
SOD that has a 50% inhi- bition on the B2/NBT system. The 50%
2
inhibition of the OH— scaveng- ing activity under 0.12 mW/cm2
blue light irradiation for 30 min was 0.97 unit SOD, which is
the optimal condition for the B2/NBT method in this study.
4. Discussion
As shown in Fig. 2, riboflavin exhibits the highest efficiency
photochemical degradation under blue light irradiation. The
effects of green, yellow and red lights on riboflavin degradation
were of low efficiency, indicating the absence of any charge
transfer inter- action between riboflavin and the phosphate
buffered solution. Ahmad et al. used a mercury vapour fluorescent
lamp (emission at 405 and 435 nm) for riboflavin photolysis. A
gradual decrease
Fig. 6. (A) Effect of one unit of SOD on OH— scavenging activity under 0.08, 0.1 and
2
0.12 mW/cm2 blue light irradiation for 10, 20 and 30 min. (B) Effect of SOD activity
on OH— scavenging activity under 0.12 mW/cm2 blue light irradiation for 10, 20 and
2
30 min. Data are represented by mean ± standard deviation, where n = 3. (For
interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
266 C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
in the absorbance of the aqueous phase at 445 nm indicated the
loss of riboflavin. On the other hand, an increase in absorbance of
a chloroform extract at 356 and 445 nm exhibited the formation
of lumichrome and lumiflavin, respectively, with time [26]. The
effectiveness of the riboflavin photolysis is mainly determined by
the wavelength of light. The photochemical degradation of ribofla-
vin may proceed through the photoreduction of the isoalloxazine
ring by electrons donated by the ribityl side chain [9].
As shown in Fig. 3, the ratio between the absorptions at 445 nm
and 373 nm indicates the efficiency of the photo-decomposition of
riboflavin. The effectiveness values of dark, Fluor-A, Fluor-B and
blue light irradiation at 1.0 mW/cm 2 for 20 min were 1.65, 1.12,
1.07 and 0.79, respectively. The lower the effectiveness value, the
higher the yield of the riboflavin photo-decomposition was. Blue
light was found to be the most efficient for the photo-
decomposition of riboflavin, and the spectra of riboflavin were
changed as a result of photo-degradation.
Human eyes are very sensitive to visible light at the wavelength
of 555 nm [27]. The colours detected by human eyes depend on
the specific wavelengths of the light sources. As shown in Table 1,
Fluor-A and Fluor-B have same radiance intensity, but the illumi-
nation intensity of Fluor-A is higher than that of Fluor-B.
At the same luminance, the IC 50 of gallic acid under Fluor-A irra-
diation at 4000 lux (0.84 mW/cm2) treatment was 32.1
lg/mL, while that under Fluor-B irradiation at 4000 lux (1.0
mW/cm2) was 53.1 lg/mL. As shown in Fig. 5(B), the IC50 of
gallic acid is 2.1-fold higher under Fluor-B than under Fluor-A
irradiation at
the same radiance intensity (1.0 mW/cm2). The scavenging capac-
ity of OH— by gallic acid is called SOD-like activity, and it can inhibit
2
the riboflavin-mediated reduction of NBT. The IC50 of gallic acid is
inversely proportional to the SOD-like activity. Hence, the SOD-like
activity of gallic acid is 2.1-fold higher under Fluor-A than
under Fluor-B irradiation at the same radiance intensity (1.0
mW/cm2).
As seen in Fig. 4, the photochemical reaction of riboflavin
was lower under Fluor-A than under Fluor-B irradiation at the
same radiance intensity. The more OH— were generated from the
2 [1] L.-S. Lai, P.-C. Chang, C.-T. Chang, Isolation and characterization of superoxide
excited riboflavin, the more antioxidants were required for the
elimination
of OH2— in the B 2/NBT method. The spectra of the fluorescent lamps
comprised multiple colour lights, as shown in Fig. 1. The emission
spectra of fluorescent lamps made by different manufacturers may
vary. Different fluorescent lamps might also generate
different amounts of OH— from the photo-decomposition of
2 [4] B. Halliwell, J.M. Gutteridge, Role of free radicals and catalytic metal ions in
riboflavin at the same irradiance or luminance. To ensure high
accuracy, the widely accepted B2/NBT assay for the quantification
of SOD activity by fluorescent lamps has to be validated.
As shown in Figs. 4 and 5(B), the photochemical reaction of
riboflavin and the IC50 of gallic acid under Fluor-A irradiation at
1.0 mW/cm2 were similar to those under blue LED irradiation at
0.1 mW/cm2, as determined by the B2/NBT method in this study.
After being light photo-energized, riboflavin is converted into
excited triplet-state riboflavin. Superoxide anion or singlet oxygen
is produced through the reaction of the excited triplet-state ribo-
flavin with triplet oxygen [8,9]. However, the quality of blue light
in the emission of the fluorescent lamps plays a major role in the
Table 1
The measurements of luminance and irradiance of fluorescence lamps and LED tube
lights.
Light source Luminance (lux)
Fluor-A 2070 4780 5940
Fluor-B 1980 4000 5340
Blue LED 350 670 910
Green LED 4560 10,190 12,790
Red LED 750 1220 1830
Irradiance (mW/cm2) 0.5 1.0 1.5
photo-decomposition of riboflavin. The effectiveness of the ribofla-
vin photolysis is controlled by the wavelength of the light source.
The photo-decomposition of riboflavin under blue light at a low
radiance intensity was selected to optimize the B 2/NBT method.
The emission spectra of coloured LEDs were always pure, clear
and in a narrow wavelength range. The blue LED light showed
the highest efficiency in the B2/NBT method.
For the same energy dose (0.144 J/cm2), the effects of one
unit of SOD on the OH— scavenging activity after blue-light
2
illumination for 30 min at 0.08 mW/cm2 and for 20 min at 0.12
mW/cm were 64.5% and 55.6%, respectively, as shown in Fig.
2
6(A). These results show that the light intensity (irradiance) has
greater influence on the photo-decomposition of riboflavin than
the irradiation time.
5. Conclusions
The effectiveness of photolysis is controlled by the wavelength
of the light source. The quality of the blue light in fluorescent
lamps is critical to the photo-decomposition of riboflavin. In this
study, blue light was found to exhibit the highest efficiency photo-
chemical reaction of riboflavin. It is concluded that the blue LED
light is better than the fluorescent lamp for the photo-
decomposition of riboflavin. Irradiation by blue light at
0.12 mW/cm2 for 30 min is determined to be the optimal condition
for the B2/NBT method.
Acknowledgment
The financial support in this work is partially from the Ministry
of Science and Technology, Taiwan, under Contracts No. MOST
103-2113-M-130-001 (Grant to L.-Y. Chen).
References
dismutase from wheat seedlings, J. Agric. Food Chem. 56 (2008) 8121–8129.
[2] L.Y. Chen, C.W. Cheng, J.Y. Liang, Effect of esterification condensation on the
Folin–Ciocalteu method for the quantitative measurement of total phenols,
Food Chem. 170 (2015) 10–15.
[3] R. Zimmermann, L. Flohe, U. Weser, H.J. Hartmann, Inhibition of lipid
peroxidation in isolated inner membrane of rat liver mitochondria by
superoxide dismutase, FEBS Lett. 29 (1973) 117–120.
human disease: an overview, Methods Enzymol. 186 (1990) 1–85.
[5] J.W. Juen, H.L. Jian, J.Y. Liang, The effect of illuminance on light induced
reduction of nitro blue tetrazolium, MC-Trans. Biotechnol. (2010).
[6] J.K. Donnelly, K.M. McLellan, J.L. Walker, D.S. Robinson, Superoxide dismutases
in foods. A review, Food Chem. 33 (1989) 243–270.
[7] W.F. Beyer Jr., I. Fridovich, Assaying for superoxide dismutase activity: some
large consequences of minor changes in conditions, Anal. Biochem. 161 (1987)
559–566.
[8] P.B. Ottaway, Stability of vitamins in food, in: The Technology of Vitamins in
Food, Chapman and Hall, London, 1993, pp. 233–244.
[9] Y. Lin, R.R. Eitenmiller, W.O. Landen, Riboflavin, in: Vitamin Analysis for the
Health and Food Sciences, CRC Press, 2008, pp. 329–360.
[10] J.-Y. Liang, C.-W. Cheng, C.-H. Yu, L.-Y. Chen, Investigations of blue light-
induced reactive oxygen species from flavin mononucleotide on inactivation
of
E. coli, J. Photochem. Photobiol., B 143 (2015) 82–88.
[11] J.-Y. Liang, J.-M.P. Yuann, C.-W. Cheng, H.-L. Jian, C.-C. Lin, L.-Y. Chen, Blue light
induced free radicals from riboflavin on E. coli DNA damage, J. Photochem.
Photobiol., B 119 (2013) 60–64.
[12] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an
assay applicable to acrylamide gels, Anal. Biochem. 44 (1971) 276–287.
[13] M. Behnamnia, K.M. Kalantar, J. Ziaie, The effects of brassinosteroid on the
induction of biochemical changes in Lycopersicon esculentum under drought
stress, Turk. J. Bot. 33 (2009) 417–428.
[14] H.-S. Kim, C.-D. Jin, Polyamines as antioxidant protectors against paraquat
damage in radish (Raphanus sativus L.) cotyledons, J. Plant Biol. 49 (2006) 237–
8240 246.
7160 [15] A. Boonmee, C. Srisomsap, A. Karnchanatat, P. Sangvanich, An antioxidant
1290 protein in Curcuma comosa Roxb. Rhizomes, Food Chem. 124 (2011) 476–480.
19,110 [16] S. Sundaram, S. Anjum, P. Dwivedi, G. Rai, Antioxidant activity and protective
2460 effect of banana peel against oxidative hemolysis of human erythrocyte at
2.0
different stages of ripening, Appl. Biochem. Biotechnol. 164 (2011) 1192–
1206.
 C.-w. Cheng et al. / Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267
[17] M. Dog˘an, Investigation of the effect of salt stress on the antioxidant
enzyme activities on the young and old leaves of salsola (Stenoptera) and
tomato (Lycopersicon esculentum L.), Afr. J. Plant Sci. 6 (2012) 62–72.
[18] C. Wang, X. Luo, Y. Tian, Y. Xie, S. Wang, Y. Li, L. Tian, X. Wang, Biphasic
effects of lanthanum on Vicia faba L. seedlings under cadmium stress,
implicating finite antioxidation and potential ecological risk, Chemosphere
86 (2012) 530– 537.
[19] H. Chen, M. Zhang, B. Xie, Components and antioxidant activity of
polysaccharide conjugate from green tea, Food Chem. 90 (2005) 17–21.
[20] S. Gangwar, V. Singh, S. Prasad, J. Maurya, Differential responses of pea
seedlings to indole acetic acid under manganese toxicity, Acta Physiol. Plant 33
(2011) 451–462.
[21] S. Verma, S.N. Mishra, Putrescine alleviation of growth in salt stressed Brassica
juncea by inducing antioxidative defense system, J. Plant Physiol. 162 (2005)
669–677.
267
[22] H.-L. Jian, C.-W. Cheng, L.-Y. Chen, J.-Y. Liang, The photochemistry of riboflavin,
MC-Trans. Biotechnol. 3 (2011) 1–11.
[23] J.M. McCord, I. Fridovich, Superoxide dismutase. An enzymic function for
erythrocuprein (hemocuprein), J. Biol. Chem. 244 (1969) 6049–6055.
[24] C. Beauchamp, I. Fridovich, Superoxide dismutase: improved assays and an
assay applicable to acrylamide gels, Anal. Biochem. 44 (1971) 276–287.
[25] A. Mariod, B. Matthä us, Y.A. Idris, S. Abdelwahab, Fatty acids, tocopherols,
phenolics and the antimicrobial effect of sclerocarya birrea kernels with
different harvesting dates, J. Am. Oil Chem. Soc. 87 (2010) 377–384.
[26] I. Ahmad, S. Ahmed, M.A. Sheraz, F.H. Vaid, Effect of borate buffer on the
photolysis of riboflavin in aqueous solution, Journal of photochemistry and
photobiology. B, Biology 93 (2008) 82–87.
[27] D. Boucar, P. Ramchandra, Light Emitting Diodes, Solar Lighting, 2011.