Journal of Photochemistry and Photobiology B: Biology 148 (2015) 262–267

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Journal of Photochemistry and Photobiology B: Biology

journal homepage: www.elsevier.com/locate/jphotobiol

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

a r t i c l e i n f o a b s t r a c t

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

1. Introduction
Superoxide dismutase (SOD) is essential to many living organ-
isms. This enzyme is one of the most important antioxidant
defence mechanisms in microorganisms and plant cells that are
exposed to oxygen [1]. The antioxidant capacity of natural ingredi-
ents is also a significant issue for foods with special dietary pur-
poses [2]. SOD, as a free radical scavenger, can convert
superoxide anion radicals (O 2 ) into H2O2 and O2 in living cells.
By scavenging O 2 , the oxidation of lipid membranes can be pre-
vented [3]. Superoxide anion radicals are intermediate products
generated during oxidation or reduction. Formed from hydroxyl
radicals or hydroxyl peroxide compounds, O 2 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
O
2 -driven reactions are more widely applied in biochemical
⇑ Corresponding author. Tel.: +886 3 3507001x3772; fax: +886 3 3593878.
E-mail address: jiyuanl@gmail.com (J.-y. Liang).
laboratories [6]. Beyer and Fridovich [7] investigated the effects
of experimental variables on two indirect assays, the xanthine oxi-
dase and cytochrome C method and the riboflavin/nitro blue tetra-
zolium method (B2/NBT). The B2/NBT method is considered simpler
and is preferred for the quantification of SOD activity in crude
extracts [7].
Riboflavin, also known as vitamin B2, is very sensitive to light. It
decomposes after being irradiated by ultraviolet (UV) or visible
light (420–560 nm) for a very short time [8], generating free radi-
cals of reactive oxygen species (ROS), such as O 2 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 O 2 . NBT reduction
causes an increase in the absorbance at 560 nm in a process that
may be inhibited by SOD. In addition, the B2/NBT method can be
employed to evaluate the contents of phenolic compounds in func-
tional foods through ROS scavenging.
In the B2/NBT method, the light sources used for generating O 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 stimulated by
ultraviolet rays to generate a hybridized visible light. However,

http://dx.doi.org/10.1016/j.jphotobiol.2015.04.028
1011-1344/Ó 2015 Elsevier B.V. All rights reserved.
 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
2 with the B2/NBT
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  106 M, 0.01 M and 1.6  104 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
O
2 , which reduced NBT to form blue formazan, which can be
detected at 560 nm (Lambda35, Perkin-Elmer).
2.5. Effects of light source on O
2 scavenging activity using gallic acid
Gallic acid was employed to determine the effects of the light
source on the O2 scavenging activity using the B2/NBT 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 O 2 generated was calculated
using the following equation, where A denotes the absorbance of
the blue formazan measured at 560 nm.
 
O
2 scavenging activityð%Þ ¼ ðAcontrol  Asample Þ=Acontrol  100%
ð1Þ
2.6. Effects of blue light on SOD activity
The effects of blue light on O
2 scavenging activity were exam-
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/cm2 for 10, 20 or 30 min. SOD activity was
defined as one unit of SOD having a 50% inhibition on the B2/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 O 2 scavenging activity with SOD (1.0 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 O

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

Fig. 2. Effects of colour-light irradiation at 1.0 mW/cm2 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/cm2 and blue light
irradiation at 0.1 mW/cm2 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
2 scavenging activity using gallic acid
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 O2 scavenging activity (%) using different levels of gal-
lic acid. As seen in Fig. 5(A), the O2 scavenging activity increased
with the addition of gallic acid and was higher under Fluor-A irra-
diation than under Fluor-B.
A characteristic concentration of chemicals, IC50, could be deter-
mined to provide 50% inhibition activity from the correlation curve
of the scavenging activity to the concentration. The IC50 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/cm2 and Fluor-B irradiation at
1.0 mW/cm2 were both insignificant.
Fig. 5. (A) O
2 scavenging activity of gallic acid under fluorescent lamp 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 O
2 scavenging activity of one unit of SOD was determined
by blue light irradiation using the B2/NBT method. As shown in
Fig. 6(A), the O
2 scavenging activity of one unit of SOD decreased
with an increase in the blue-light irradiation time for the photo-
chemical treatment.
The effect of the SOD activity on O
2 scavenging activity under
blue-light irradiation were investigated using the B2/NBT method.
As shown in Fig. 6(B), the O2 scavenging activity increased with
the SOD activity, and the 50% inhibition value of the O
2 scavenging
activity at 0.12 mW/cm2 blue-light irradiation 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% inhibition of the O2 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 O2 scavenging activity under 0.08, 0.1 and
0.12 mW/cm2 blue light irradiation for 10, 20 and 30 min. (B) Effect of SOD activity
on O 2
2 scavenging activity under 0.12 mW/cm blue light irradiation for 10, 20 and
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/cm2 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 IC50 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 O
2 by gallic acid is called SOD-like activity, and it can inhibit
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 O 2 were generated from the excited
riboflavin, the more antioxidants were required for the elimination
of O
2 in the B2/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 O 2 from the photo-decomposition of 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 B2/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 O2 scavenging activity after blue-light illumination
for 30 min at 0.08 mW/cm2 and for 20 min at 0.12 mW/cm2 were
64.5% and 55.6%, respectively, as shown in Fig. 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).
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