Photo-oxidation of some flavonoids with photochemically generated t-BuO? radicals in a t-BuOH water system using a kinetic approach

Article abstract of DOI:10.1002/jccs.201700342

As a dietary component, flavonoids are thought to have a variety of pharmacological and health-promoting properties in both in vivo and in vitro systems. This variety is attributed to their high antioxidant capacity, which in turn is associated with their free radical scavenging properties. In order to understand the mechanism of t-BuO^? radical scavenging properties of some flavonoids, viz, quercetin (QU), apigenin (AP), daidzein (DA), genistein (GE), myricetin (MY), and kaempferol (KA), a kinetic study of photo-oxidation of these flavonoids with photochemically generated t-BuO^? radicals in a t-BuOH water (2:1 v/v) system was carried out. The oxidation of flavonoids by t-BuO^? radicals was performed spectrophotometrically by measuring the absorbance of quercetin (375 nm), apigenin (340 nm), daidzein (250 nm), genistein (263 nm), myricetin (255 nm), and kaempferol (375 nm) at their respective λ_max. The initial rates of oxidation of flavonoids increased with [flavonoid], [t-BuOOH], and light intensity. The quantum yields (φ) were considered from the initial rate of oxidation of flavonoids by t-BuO^? radicals and measured light intensity at 254 nm. The order on [flavonoid] and [t-BuOOH] was found to be fractional, whereas the order on light intensity was found to be one. The products of the oxidation of flavonoids by t-BuO^? radicals were identified using LC–MS and FTIR analysis.

Full text of DOI:10.1002/jccs.201700342

Received: 5 October 2017
Revised: 15 March 2018
Accepted: 22 April 2018
DOI: 10.1002/jccs.201700342
A R T I C L E
Photo-oxidation of some flavonoids with photochemically
•
generated t-BuO radicals in a t-BuOH water system using
a kinetic approach
1
2
3
Radhika Mallepu | Laxmi Potlapally | Vijaya Lakshmi Gollapalli
1
Department of Chemistry, University College of
As a dietary component, flavonoids are thought to have a variety of pharmacologi-
cal and health-promoting properties in both in vivo and in vitro systems. This vari-
ety is attributed to their high antioxidant capacity, which in turn is associated with
their free radical scavenging properties. In order to understand the mechanism of
Science, Osmania University, Hyderabad, India
2
Department of Pharmacy, University College of
Technology, Osmania University, Hyderabad,
India
3
Department of Chemistry, University College for
•
t-BuO radical scavenging properties of some flavonoids, viz, quercetin (QU), api-
Women, Osmania University, Hyderabad, India
genin (AP), daidzein (DA), genistein (GE), myricetin (MY), and kaempferol (KA),
a kinetic study of photo-oxidation of these flavonoids with photochemically gener-
Correspondence
Vijaya Lakshmi Gollapalli, Department of
Chemistry, University College for Women,
Osmania University, Koti, Telangana State,
Hyderabad 500 095, India.
•
ated t-BuO radicals in a t-BuOH water (2:1 v/v) system was carried out. The oxi-
•
dation of flavonoids by t-BuO radicals was performed spectrophotometrically by
measuring the absorbance of quercetin (375 nm), apigenin (340 nm), daidzein
Email: gvjlakshmi@gmail.com
(250 nm), genistein (263 nm), myricetin (255 nm), and kaempferol (375 nm) at
Funding information
University Grants Committee, Grant/Award
Number: UGC-BSR Start-up Research Grant,
Lr. No. F.30-61/2; UGC-BSR Start-up Research
Grant, Grant/Award Number: F.30-61/2014(BSR)
their respective λmax. The initial rates of oxidation of flavonoids increased with
[
flavonoid], [t-BuOOH], and light intensity. The quantum yields (φ) were consid-
•
ered from the initial rate of oxidation of flavonoids by t-BuO radicals and mea-
sured light intensity at 254 nm. The order on [flavonoid] and [t-BuOOH] was
found to be fractional, whereas the order on light intensity was found to be one.
•
The products of the oxidation of flavonoids by t-BuO radicals were identified
using LC–MS and FTIR analysis.
KEYWORDS
•
flavonoids, oxidation products, photo-oxidation, quantum yields, t-BuO radicals
[
2,3]
1
| INTRODUCTION
diseases, cancers, and other age-related diseases.
Recent
interest in these substances has been stimulated by the medi-
cal efficacies arising from the antioxidant activities of these
polyphenolic compounds.
Flavonoids are a large group of polyphenolic compounds
with a benzo-γ-pyrone structure. Structurally, flavonoids are
based on a 15-carbon skeleton consisting of two benzene
rings (A and B) linked via a heterocyclic pyrane ring
[
4–6]
The chemical properties of flavonoids depend on their
structure, the degree of hydroxylation, other substitutions
[1]
[3]
(C) (Figure 1). Structural variations within the rings subdi-
and conjugations, and the degree of polymerization. It was
vide the flavonoids into several families, viz, flavonols, fla-
vones, flavanols, isoflavones, catechins, anthocynidins, etc.
A multitude of studies has suggested protective effects of
flavonoids against many infectious (bacterial and viral dis-
eases) and degenerative diseases such as cardiovascular
suggested that the configuration, substitution, and an overall
number of hydroxyl groups noticeably affect some of the
mechanisms of antioxidant activity, such as radical scaveng-
ing and metal ion chelation ability. The B ring hydroxyl
configuration is the greatest significant contributing factor of
[
7]
©
2018 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2018;1–8.
http://www.jccs.wiley-vch.de
1

2
MALLEPU ET AL.
and DNA, affect cell membrane integrity, and result in cell
[
18,19]
injury in hepatocytes and rat liver.
Previous studies on
the reactivity of tert-butoxyl radicals propose that these spe-
cies mutually influence the anticipated attack on the base and
[
20,21]
the sugar moieties of DNA.
However, not many studies
•
are available highlighting the alkoxyl (t-BuO radicals) radical
scavenging activities of flavonoids. It is in this background
that we have chosen tert-Butyl hydroperoxide (t-BuOOH) as
the model peroxide in the present study, which results in
•
•
FIGURE 1 General structure of flavonoids
t-BuO and OH radicals on homolysis.
Although many structure–activity relationship (SAR)
investigations have been performed on the antioxidant
activity of flavonoids, only a few studies are available that
scavenging of reactive nitrogen species (RNS) and reactive
oxygen species (ROS) because of its donation of hydrogen
and an electron to hydroxyl, peroxyl, and peroxynitrite radi-
cals, and the subsequent radicals produced can be deloca-
•
focus on the kinetic model to assess the t-BuO radical
scavenging abilities of flavonoids. Thus, a systematic
mechanistic investigation has been conducted to focus on
the interaction of some flavonoids (Figure 2), viz, quercetin
[8]
lized over the flavonoid structure. Various mechanisms of
antioxidant action by flavonoids were suggested, of which
scavenging ROS is considered one of the most prominent.
Flavonoids that are powerful antioxidants possess lower
redox potentials and thus are thermodynamically able to
reduce highly oxidizing free radicals, such as superoxide,
peroxyl, alkoxyl, and hydroxyl radicals, mainly by hydrogen
(
QU), apigenin (AP), daidzein (DA), genistein (GE), myri-
•
cetin (MY), and kaempferol (KA), with t-BuO radicals
using a kinetic approach. Particular attention is paid to
SAR studies of six flavonoids regarding their chemical
structure and radical scavenging properties. An effort has
also been made to suggest a likely mechanism for oxidation
[
9,10]
atom donation.
Manifestation, position, structure, and a
•
of quercetin by t-BuO radicals based on the kinetic results
total number of sugar moieties in flavonoids (flavonoids gly-
cosides) play a major role in antioxidant activity. The mech-
anisms accountable for these effects are not clear but appear
to be related to their antioxidant effect and ability to scav-
enge free radicals, which requires further investigation.
Alkoxyl radicals are produced on UV-A irradiation or
and the products identified using LC–MS and FTIR analy-
sis. Such studies have immense significance in understand-
ing the molecular mechanisms underlying the synergistic
interactions and help in exploring the possible role of fla-
vonoids in cooperative (synergistic) interactions with other
phytochemicals.
[
11,12]
metal-catalyzed decomposition of lipid hydroperoxides.
They have been reported to induce strand breaks in super-
[13]
coiled pBR322 DNA and may play a significant role in the
2 | MATERIALS AND METHODS
[14]
tumor-promoting activity of peroxides.
Strand break for-
Quercetin, apigenin, daidzein, genistein, myricetin, and
kaempferol were purchased from Sigma-Aldrich Chemical
Co., St. Louis, MO, and used as received. All solutions were
prepared fresh using double-distilled water. t-BuOOH was
used as received from Merck-Schuchardt of Germany and
mation and base oxidation have been observed as necessary
modifications induced by the alkoxyl radicals in DNA dam-
[15]
age. Tert-Butyl hydroperoxide (t-BHP) is a recognized oxi-
dant that has been used as a model oxygen-centered radical
for the study of mechanisms of oxidative cell injury in mam-
malian cells and to investigate mechanisms of its interaction
[
22]
was estimated through the iodometric method.
The irradi-
[
16,17]
ations were performed at ambient temperature in a quantum
yield reactor model QYR-20 supplied by Photophysics,
England, attached to a 400 W, medium-pressure, mercury
lamp. The quartz cuvette containing the sample was irradi-
ated, the irradiations were interrupted at specific intervals of
time, and the absorbance was noted. The light intensity
with polyphenols.
These free radicals readily cross cellu-
lar membranes and induct lipid peroxidation, damage protein
OH
OH
OH
OH
HO
O
HO
HO
O
O
equivalent to the irradiating wavelength (254 nm) was mea-
OH
OH
[23]
O
OH
O
OH
O
OH
sured using peroxydisulphate chemical actinometry.
On
Quercetin
Kaempferol
Apigenin
photolysis, t-BuOOH was triggered at 254 nm to make OH
and t-BuO radicals through the homolytic cleavage of –O–
O– bond. The OH radicals produced were scavenged by a
OH
OH
OH
HO
O
HO
O
HO
O
•
OH
sufficient concentration of t-BuOH present in the solvent
mixture.
OH
O
O
OH
O
[24]
OH
OH
Geninstein
Daidzein
Myricetin
The kinetic path of the reaction mixture enclosing flavo-
FIGURE 2 Structures of flavonoids studied
noid and t-BuOOH in t-BuOH–water (2:1 v/v) ratio, taken in

MALLEPU ET AL.
3
a specially designed path length of the quartz cuvette, is
3 | RESULTS AND DISCUSSION
1
cm, proper for both irradiations and absorbance determi-
nations. The reaction mixture was purged with N before
Flavonoids and their glycosides were found to perform as
hydrophilic antioxidants, antimicrobials, photoreceptors,
visual attractors, feeding repellents, and as UV-light filters
and substrate for polyphenol oxidases. It is reported that
flavonoids might account for a reasonable part of the health
benefits associated with vegetable and fruit consump-
2
irradiation. The absorbance measurements were taken at
the max of flavonoid on a Systronics UV–Visible Double-
beam spectrophotometer (model 2202). It is known that
t-BuOOH is activated by the radical reaction of the absorp-
tion of light at 254 nm. However, some of the substrates
may have strong absorption in this region. However, in the
absence of t-BuOOH in the reaction mixture, they did not
undergo any noticeable chemical change on shining the
light. The light intensity at 254 nm was used to determine
the quantum yields of oxidation of antioxidants under dif-
ferent experimental conditions. The initial rate of photo-
oxidation of the flavonoid by t-BuOOH in a solvent
mixture t-BuOH–water (2:1 v/v) ratio was calculated from
the plots of absorbance versus time using the Microcal Ori-
gin (version 6.0) software program. The products of oxida-
tion of flavonoids by t-BuO radicals were identified using
Liquid Chromatography-Mass Spectrometry (LC–MS) and
Fourier Transform Infrared (FTIR) analysis. For the prod-
uct analysis, about 0.01 M of flavonoid solution in
t-BuOH–water (2:1 v/v) ratio added to 0.1 M t-BuOOH
solution was taken in a quartz cuvette and irradiated for
[
25,26]
tion.
The antioxidant nature of flavonoids is attrib-
uted to the effective ROS scavenging of radicals. This is
due to their low redox potentials (0.2 < E < 0.8) depend-
o
ing on the arrangement of functional groups about the
[27,28]
nuclear structure.
The free radical scavenging poten-
tial of flavonoids has been related to the stability of their
radical species and is expected to increase by extended
conjugation. Antioxidant capacity assays have mainly clas-
sified flavonoids as scavenging by electron transfer (ET) or
[29]
by hydrogen atom transfer (HAT).
•
The photo-oxidation of flavonoids by t-BuO radicals
was carried out under different experimental conditions. The
initial rates of oxidation of flavonoids were found to increase
with an increase in [flavonoid], [t-BuOOH], and light
strength in all the cases and are given in Tables 1 and 2. The
quantum yields (φ) were estimated from the initial rate of
4
8 hr. The resultant solution was used for FTIR analysis
and LC–MS.
The FTIR Spectrophotometer (Shimadzu IR Prestige21
•
oxidation of flavonoids by t-BuO radicals and measured
light intensity at 254 nm, the wavelength at which t-BuOOH
•
•
was activated to give t-BuO and OH radicals. The quan-
Instrument) was used to record the FTIR spectra of a photo-
oxidized mixture of the flavonoid with the t-BuOOH solu-
tion through the KBr pellet method. The scan was carried
tum yields of all the flavonoids were found to depend on
[flavonoid] and [t-BuOOH] and were independent of light
−
1
intensity and are given in Tables 3 and 4. The order on [fla-
vonoid] and [t-BuOOH] was found to be fractional, whereas
the order on light intensity was found to be one. The absorp-
tion spectra for the photo-oxidation of the flavonoids studied
out in the range of 250–4000 cm . LC–MS spectra of the
samples were recorded using Shimadzu on column Ascentis
Express C-18 (50 × 3.0 mm, 2.7 um), a mobile phase of
acetonitrile +5% 0.025% aqueous trifluoroacetic acid with a
flow rate of 1.2 mL/min (Gradient) connected to MS, with
the ESI analyzer in positive/negative ion mode.
•
with t-BuO radicals at different time intervals are given in
Figure 3.
•
TABLE 1 Effect of [flavonoid] and [t-BuOOH] on the rate of oxidation of flavonoid by t-BuO radicals in t-BuOH–water (2:1 v/v) system. Light
1
5
intensity = 5.7549 × 10 quanta/s, pH ~ 7.5, room temperature = 298 K
Rate × 10 dm mol s−1
9
3
−1
Concentration
Flavonoid] × 10 mol/dm3
6
[t-BuOOH] × 10 mol/dm3
3
MY
QU
KA
AP
GE
DA
[
1
2
3
5
8
.0
.0
.0
.0
.0
5.0
5.0
—
11.944
16.527
19.097
27.083
—
11.178
15.921
18.834
26.578
32.655
39.837
49.322
—
—
0.3274
0.3741
—
0.4150
0.4603
—
0.5069
—
0.2807
—
5.0
5.0
1.0138
1.7778
2.9166
6.1458
12.826
—
0.5526
0.8157
1.2543
2.0438
3.3508
—
0.6055
0.9330
1.9333
2.3505
—
0.6238
1.1507
2.0079
2.6984
—
5.0
1
2
4
0.0
0.0
0.0
5.0
42.632
50.000
—
5.0
5.0
1.0
1.0
1.0
1.0
5.5556
9.027
22.564
—
—
—
—
2.0
—
—
—
—
—
10.0
10.0
15.0
—
—
—
—
—
1
0.0
0.0
5.3889
6.6944
47.967
57.310
2.0350
2.2807
2.1620
2.4851
4.3650
5.7143
1
—

4
MALLEPU ET AL.
TABLE 2 Effect of light intensity on the rate of oxidation of flavonoid
feature for the activity as it stabilizes the radical formed at the
ring B. If there are no hydroxyl groups on ring B, then the
hydroxylation of ring A becomes important.
•
by t-BuO radicals in t-BuOH–water (2:1 v/v) system.
−
3
3
−6
3
[
t-BuOOH] = 5.0 × 10 mol/dm , [flavonoid] = 10.0 × 10 mol/dm
[2,32,33]
−
6
3
and [QU] = 1.0 × 10 mol/dm , pH ~ 7.5, room temperature = 298 K
Some of the SAR studies on polyphenolic compounds
indicated that QU has a higher antioxidant capacity com-
Rate × 10 dm mol s−1
9
3
−1
Light intensity × 10−15
quanta/s
MY
QU
KA
AP
GE
DA
[34–36]
[37]
pared to the other polyphenols.
Yang et al. studied
8
5
3
.4379
.7549
.2375
5.0000 12.500 49.783 2.5614 2.6959 2.7778
2.9166 11.944 39.837 1.2543 1.9333 2.0079
1.7778 10.069 28.945 0.7894 1.0003 1.0317
the antioxidant activities of flavonoids against lard oil oxida-
tion using the Rancimat test. Based on the calculated protec-
tion factor (PF) values, the order of antioxidant activity was
found to be QU > KA > DA ~ GE > AP. The antioxidant
Previous studies on the reactions of flavonoids with free
performance of flavonoids was explained by both ΔH (vari-
f
radicals have correlated the number and position of hydroxyl
groups and the extension of conjugation to the efficiency of
ance in the heat of formation of the phenolic antioxidant and
its free radical produced after H-abstraction) and the several
numbers of hydroxyl groups in the conjugated B and C ring
[27–30]
flavonoids as antioxidants.
Three structural requirements
[
38]
were found to be important: (a) the ortho-dihydroxy (catechol)
structure in the B ring; (b) the 2,3-double bond, in conjugation
with the 4-oxo function; and (c) the presence of both 3- and
system. Burda and Olezek studied antioxidant and antiradi-
cal activities of flavonoids and found similar results wherein
QU and KA showed higher antioxidant activity. Chung
[
39]
5
-OH groups. Apart from these structural requirements, the
et al.
also predicted similar results from their DPPH free
number and position of hydroxyl substituents on the flavonoid
molecule, the existence of glycosides, and the overall degree
of conjugation are important in determining their activities. A
common flavonoid that meets the above three criteria is QU,
showing the highest antioxidant capacity. Based on a low
dipole moment, quantum mechanical calculations and a signifi-
cant deviation from the A-to-B dihedral angle are necessary
radical and inhibition of TPA-induced free radical formation
[
27]
with cultured HL-60 cells studies. Jovanovic et al. proposed
that the desirable electron-donating properties originate from
the electron-donating hydroxy group in the C ring, which is
conjugated to the catechol (B ring) radical through the
[
40]
2,3-double bond. The kinetic results of Lin et al. have indi-
cated that the conjugation between the pyrone and catechol
rings play an important role in the reactivity of flavonoids.
However, the effectiveness of an antioxidant is determined by
[31]
for high antioxidant efficiency for flavonoids.
It has been
indicated that the hydroxylation of ring B is the most important
•
TABLE 3 Effect of [flavonoid] and [t-BuOOH] on the quantum yield of oxidation of flavonoid by t-BuO radicals in t-BuOH–water (2:1 v/v) system. Light
1
5
intensity = 5.7549 × 10 quanta/s, pH ~ 7.5, room temperature = 298 K
Concentration Quantum yield (φ)
Flavonoid] × 10 mol/dm
6
3
3
3
[
[t-BuOOH] × 10 mol/dm
MY
QU
KA
AP
GE
DA
1
2
3
5
8
1
2
4
1
1
1
1
1
.0
5.0
5.0
—
0.00375
0.00518
0.00599
0.00850
—
0.00350
0.00499
0.00591
0.00834
0.01025
0.01250
0.01548
—
—
0.00010
0.00012
—
0.00013
0.00014
—
.0
0.00015
—
0.00008
—
.0
5.0
.0
5.0
0.00031
0.00055
0.00091
0.00192
0.00402
—
0.00017
0.00025
0.00045
0.00064
0.00105
—
0.00019
0.00029
0.00060
0.00073
—
0.00019
0.00036
0.00063
0.00084
—
.0
5.0
0.0
0.0
0.0
.0
5.0
0.01338
0.01569
—
5.0
5.0
1.0
0.00174
0.00283
0.00708
—
—
—
—
.0
2.0
—
—
—
—
—
.0
10.0
10.0
15.0
—
—
—
—
—
0.0
0.0
0.00169
0.00210
0.01506
0.01799
0.00063
0.00071
0.00067
0.00078
0.00137
0.00179
—
•
−3
3
TABLE 4 Effect of light intensity on the rate of oxidation of flavonoid by t-BuO radicals in t-BuOH-water (2:1 v/v) system. [t-BuOOH] = 5.0 × 10 mol/dm ,
[
flavonoid] = 10.0 × 10− mol/dm and [QU] = 1.0 × 10 mol/dm , pH ~ 7.5, room temperature = 298 K
6
3
−6
3
Quantum yield (ϕ)
Light intensity × 10−15 quanta/s
MY
QU
KA
AP
GE
DA
8
5
3
.4379
.7549
.2375
0.00107
0.00091
0.00099
0.00392
0.00375
0.00314
0.01066
0.01250
0.01615
0.00054
0.00045
0.00044
0.00057
0.00060
0.00055
0.00059
0.00063
0.00057

MALLEPU ET AL.
5
[
44–47]
investigations.
In the present study, MY showed anti-
oxidant activity toward t-BuO radicals that was higher than
AP but lower than other flavonoids studied. MY has the
largest number of phenolic OH groups among all the six fla-
vonoids studied but exhibits poor activity in scavenging t-
•
BuO radicals. According to Rice-Evans et al., the presence
of a third OH group in the B ring situated in ortho position
to other OH groups in the B ring, as in MY does, not
improve the effectiveness of flavonoids against aqueous
[
6]
phase radicals.
GE and DA are the two most important isoflavones con-
tained in about equal quantities in soy and are phytoestro-
[
48–51]
gens. Accumulating evidence
from in vivo studies
suggests that isoflavones may have an impact on cardiovas-
cular diseases, cancers, and osteoporosis and that their anti-
oxidant activity might be responsible for their protective
FIGURE 3 Absorption spectra of photo-oxidation of six flavonoids in the
presence of t-BuOOH at different irradiation times in t-BuOH–water (2:1 v/v)
•
effects. GE and DA were found to have comparable t-BuO
medium at their corresponding λmax[flavonoid] = 2.0 × 10− mol/dm ,
5
3
radical scavenging capacity in the present study. Similar
−
3
3
[
t-BuOOH] = 5.0 × 10 mol/dm , pH 7.5, room temperature = 298 K, light
[52]
results were also reported by Foti et al. , indicating that
1
5
intensity = 5.7549 × 10 quanta/s. KA, kaempferol; MY, myricetin; AP,
apigenin; GE, genistein; DA, daidzein and QU, quercetin
DA was just as effective as GE against induced oxidative
damage in Jurkat T-cell line and peripheral blood lympho-
cytes of healthy subjects. This may be due to the structural
similarities between DA and GE, the presence of phenolic
many factors, including its activation energy, rate constant,
oxidation-reduction potential, bond dissociation energies
0
0
moiety at 5 position of the B ring instead of 4 position as
(BDE), the stability of the radical intermediate, thermal stabil-
[
31,34]
observed in other flavonoids studied.
Another notable
ity, and hydrophobicity. In the present kinetic study, the order
of the reactivity of flavonoids with t-BuO radicals was found
to be QU > KA > MY > DA > GE > AP.
feature that can be observed is that the presence of 5-OH
group on the A ring did not affect the antioxidant capac-
ity of GE.
QU, a plant-derived aglycone form of flavonoid glyco-
sides, satisfies all the structural features required to show
powerful free radical scavenging ability and hence counter-
acts oxidative stress generated as a result of ROS, which
contributes to the genesis of atherosclerosis, nervous disor-
AP is one of the active ingredients found in fruits and
vegetables and an outstanding food functional factor and
[
53]
potential therapeutic drug for some diseases.
It has been
widely investigated for its anti-inflammatory effects, free
radical scavenging effects, and growth inhibitory properties
[
41]
ders, obesity, etc.
In our kinetic study, QU was found to
[
54,55]
in several cancer lines.
Recent clinical studies demon-
have the highest antioxidant property among all the six fla-
vonoids studied.
strated that the AP intake was associated with a suggestive
[
56]
decrease in woman's risk of ovarian cancer.
The least t-
KA is a flavonoid found in many edible plants and is
commonly used in traditional medicine. Numerous preclini-
cal studies have shown that KA has a broad variety of phar-
macological activities, including antioxidant, antimicrobial,
anticancer, cardioprotective, anti-osteoporotic, and antialler-
•
BuO radicals scavenging activity was observed for AP in
the present study. Two hydroxyl groups in the B ring are
required to obtain a stronger antioxidant potential; a lack of
one of them in AP significantly reduced this activity. Similar
[
57]
[
42,43]
results were obtained by Majewska et al.,
who evaluated
gic activities.
KA was found to demonstrate slightly
•
the free radical scavenging activity of flavonoids through
their ability to quench the synthetic 2,2- diphenyl-
1-picrylhydrazyl (DPPH) radical and found that AP had the
weakest radical scavenging potential against DPPH.
less antioxidant activity toward t-BuO radicals compared to
QU but was higher than other flavonoids in our present
study. One of the possible reasons could be the presence of
the 4-OH group in B ring instead of the catechol structure
present in QU, which might have decreased its antioxidant
power toward scavenging t-BuO radicals.
Flavonoids, in general, undergo photo-oxidation, with t-
BuO• radicals producing the corresponding semiquinone
radical (phenoxyl radical), which combined with phenoxy or
other radicals available in the medium to give either dimer
or other stable final products. An analysis of the potential
Compared to QU, MY is found in less common foods,
viz, black currants, black grapes, cranberries, broad beans,
etc, and is consequently consumed to a lesser extent. Most
studies on the antioxidant activity of MY have shown that it
is more efficient than QU as an antioxidant in oils, emul-
sions, and LDL oxidation, whereas the reverse order has also
been found for oxidation in emulsions and few other
•
products of oxidation of flavonoids by t-BuO radicals was
carried out using FTIR analysis and LC–MS of the irradiated
reaction mixture. In the FTIR spectra of all the flavonoids,
two strong peaks were observed: one in the region of

6
MALLEPU ET AL.
−1
1
600–1700 cm , indicating the presence of carbonyl group
formed due to the oxidation of –OH group to semiquinone or
−1
a quinone, and a broad peak in the region of 3000–3500 cm ,
representing H-bonding of the phenolic groups of the polyphe-
nols studied. The FTIR spectrum of the photo-oxidation of
QU, as a representative compound, has been given in Figure 4,
showing these peaks and clearly indicating the formation of
corresponding quinones as one of the possible oxidation prod-
ucts. The LC–MS chromatogram of the oxidized mixture of
the flavonoids have indicated the formation of the numerous
oxidation products at different retention times (RT), which has
made the analysis more complicated, but attempts were made
to identify few oxidation products from the complex mixture
for QU as given in Figure 5.
The various classes of flavonoids were found to change
in the level of oxidation and arrangement of substitution of
the C ring, while specific compounds within a class vary in
[1,2]
the pattern of substitution of the A and B rings.
Oxida-
tion studies of quercetin, which is one of the most commonly
found flavonoids in plant extracts, is well documented. Few
studies have investigated the degradation of quercetin in the
dark, when dissolved in an alkaline medium, treated with
•
FIGURE 5 LC–MS of QU photo-oxidized by t-BuO radicals in t-BuOH–
water (2:1 v/v) ratio
[58–60]
radical generators, or electrochemically oxidized.
Zare
the positive ion mode. The increase in the absorbance at
05 nm in the UV spectrum may be attributed to the quinone
[
61]
et al.
studied electrochemical behavior of quercetin using
3
cyclic voltammetry, chronoamperometry, rotating disk elec-
trode voltammetry, and quantum mechanical calculations
and suggested that the o-quinone derived from quercetin par-
ticipates in Michael addition reaction with quercetin and pro-
duces a dimer.
formed (Figure 3). Previous studies on the oxidation of QU
indicated the formation of quinone in which the two –OH
[
64,65]
groups in ring B are readily oxidized.
It has recently
been reported that polyphenol oxidase (PPO) and mushroom
tyrosinase can oxidize QU to four corresponding tautomeric
[62,63]
[66]
A significant amount of literature
is available on oxi-
forms of quinones. Timbola et al. studied oxidation of QU
dation of flavonoids by radicals, and a kinetic approach has
been used here to investigate the oxidation of flavonoids by
t-BuO radicals. In the present study, the phenoxyl (semiqui-
by cyclic voltammetry, controlled-potential electrolysis,
and UV–Vis spectroscopy. The two-electron, two-proton
oxidation process of QU at the first peak directed to the
materialization of the conforming ortho-quinone, which is
an electrochemically active and unstable species. Xu
•
none) radical of QU was observed in LC–MS at an RT of
2.467–2.533 min by the loss of proton from 4’-OH of the B
[
67]
ring, which further loses another proton from 3’-OH and gives
quinone with m/z of 300.8 at an RT of 2.567–2.617 min in
et al.,
in their in situ spectroelectrochemical study of QU
oxidation, reported the formation of four possible quinones
and their structures. The differential pulse voltammograms
of QU showed that the first oxidation peak, with peak
potential E = + 0.30 V, corresponds to the loss of two
p
electrons and two protons from ring B, a reversible process
giving o-quinone species, which can undergo rearrangements
to give other products. About 32% (relative amount) of a
dimer of QU was detected at an RT of 3.100–3.183 min with
m/z of 603 in positive ion mode, for which the possible mech-
[
68]
anism is discussed in Figure 6. Chervyakovsky et al.
also
indicated the formation of dimer as one of the major products
in the oligomeric oxidation of QU due to the possible random
cyclization of QU leading to dimers and trimers. Es-Safi
[
69]
et al.
have studied the enzymatic oxidation of flavonols
and indicated the formation of dimers for a few of them
through mass spectral analysis and reported that QU allowed
the formation of two pseudodimeric compounds at m/z
•
FIGURE 4 FTIR of QU photo-oxidized by t-BuO radicals in t-BuOH–
water (2:1 v/v) ratio
[
58]
498 and 618. Krishnamachari et al.
also indicated the

MALLEPU ET AL.
7
•
nature toward t-BuO radicals. The product analysis showed
the formation of phenoxyl radical such as semiquinone for
all the flavonoids studied, which may further combine with
other species available to form a dimer or other oxidation
products. The formation of an intramolecular hydrogen bond
between hydroxyl groups of flavonoids increases the stabil-
ity of the phenoxyl radicals. The fate of the phenoxyl radi-
cals depends on their structure and the presence of species
affecting the stability of the free radicals. However, it is
tough to clearly establish the pathway for the formation of
these comprehensive oxidation products of flavonoids
because an antioxidant decreases the oxidative stress at low
concentrations. As a consequence, further research should be
performed to convincingly address the importance of flavo-
noids as free radical scavengers. Such studies will also throw
light on the understanding of synergistic interactions of these
molecules with other phytochemicals forming an integral
part of the diet.
ACKNOWLEDGEMENTS
The corresponding author is thankful to the University
Grants Commission (UGC), New Delhi, for funding the pre-
sent work through UGC-BSR Start-up Research Grant,
Lr. No. F.30-61/2014(BSR) dated 18.9.2014. The authors
are also thankful to Dr P. Someswar, Department of Chemis-
try, Nizam College, Osmania University, Telangana State,
India, for useful suggestions.
FIGURE 6 Reaction scheme for the oxidation of QU by photochemically
generated t-BuO radicals showing one of the possible quinone and dimer
as oxidation products
•
formation of the dimeric product along with other oxidation
products in the oxidation of flavonoids by the peroxyl radical
generator 2,2 -azobis-isobutyronitrile (AIBN), which were
ORCID
Vijaya Lakshmi Gollapalli
http://orcid.org/0000-0001-7183-4908
0
identified by chromatographic techniques and acknowl-
edged by NMR and MS analyses. At RTs of 2.467–2.533
and 2.567–2.617, the two peaks observed in the range of
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How to cite this article: Mallepu R, Potlapally L,
[
[
[
Gollapalli VL. Photo-oxidation of some flavonoids
•
with photochemically generated t-BuO radicals in a
t-BuOH water system using a kinetic approach.
J Chin Chem Soc. 2018;1–8. https://doi.org/10.1002/
jccs.201700342
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1