Antiradical and reductant activities of anthocyanidins and anthocyanins, structure-activity relationship and synthesis

Article abstract of DOI:10.1016/j.foodchem.2015.09.003

Eight anthocyanidins, seven anthocyanins and two synthesized 4′-hydroxy flavyliums were examined as hydrogen donors to DPPH, ABTS and hydroxyl radicals, and as electron donors in the FRAP assay. Most compounds gave better activities than trolox and catech

Full text of DOI:10.1016/j.foodchem.2015.09.003

Food Chemistry 194 (2016) 1275–1282
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Antiradical and reductant activities of anthocyanidins and anthocyanins,
structure–activity relationship and synthesis
b
b
Hussein M. Ali a, , Wafaa Almagribi , Mona N. Al-Rashidi
⇑
a Agricultural Biochemistry Department, Faculty of Agriculture, Ain Shams University, Shoubra El-Kheima, Cairo, Egypt
b Chemistry Department, Faculty of Science for Girls, Dammam University, Dammam, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Eight anthocyanidins, seven anthocyanins and two synthesized 40-hydroxy flavyliums were examined as
hydrogen donors to DPPH, ABTS and hydroxyl radicals, and as electron donors in the FRAP assay. Most
compounds gave better activities than trolox and catechol. A structure–activity relationship (SAR) study
showed that, in the absence of the 3-OH group, radicals of the 4, 5 or 7-OH groups can only be stabilized
by resonance through pyrylium oxygen, while 3-OH group improved hydrogen atom donation because of
the stabilization by anthocyanidin semiquinone-like resonance. Electron donation was also enhanced by
the 3-OH group. Both anthocyanidins and their respective anthocyanins showed similar trends and close
activities. Different types of sugar unit bonded to the 3-OH group or counter ion had minor effect on
activities. The catechol structure improved both hydrogen and electron donation. Compounds lacking
the catechol structure had a decreasing order of H-atom and electron donation (Mv > Pn > Pg > Ap >
40-OH-flavylium) consistent with the decreasing number of their hydroxyl and/or methoxy groups.
Ó 2015 Elsevier Ltd. All rights reserved.
Article history:
Received 28 March 2015
Received in revised form 21 July 2015
Accepted 2 September 2015
Available online 3 September 2015
Keywords:
Anthocyanins
Antioxidants
SAR
Radical stabilization
Semiquinone resonance
1. Introduction
platelet aggregation, cardiovascular complication, liver damage,
lipid peroxidation and cancer tumor growth (Kong et al., 2003).
Anthocyanins are glycosides of aglycons called anthocyanidins;
both forms are well distributed in the plant kingdom especially in
vegetables, fruits and flowers, and are responsible for many of their
colors, particularly orange, red, blue and purple. Anthocyanidins
are an interesting class of flavonoids characterized by having an
oxonium ion, namely 2-phenylbenzopyrylium (flavylium). There
are 23 known anthocyanidins and more than 500 anthocyanins
that occur naturally. The most common anthocyanidins in plants
are cyanidin (Cn), pelargonidin (Pg), peonidin (Pn), delphinidin
(Dp), petunidin (Pt) and malvidin (Mv); they vary, as in other flavo-
noid groups, in their number, and spatial distribution, of hydroxyl
Many of these biological activities have been attributed to the
potent antiradical and antioxidant activity of anthocyanins. There
are two widely accepted mechanisms for radical scavenging activ-
ity of phenolic compounds, the hydrogen-atom transfer mecha-
nism (where the antioxidant donates a hydrogen atom to the
active radical and gives a stable phenoxyl radical in one step)
and the single electron transfer mechanism (where an electron
and a proton are transferred in two consecutive steps to give first
a radical cation then the phenoxyl radical, respectively). Both
mechanisms are thought to work as parallel reactions (Klein &
Lukes, 2007; Huang, Boxin, & Prior, 2005).
ˇ
and methoxy groups (Castaneda-Ovando, Pacheco-Hernández,
The antioxidant and antiradical activity of anthocyanidins and
anthocyanins are strongly related to their structural features
including the kind, number and position of substituents on the fla-
vylium ion. The number and position of hydroxyl and methoxy
substituents, as electron donating groups, were found to have a
great impact on the antioxidant activity of anthocyanidins
(Azevedo et al., 2010; Kähkönen & Heinonen, 2003). Anthocyani-
dins with catechol (1,2-diphenol) or pyrogallol (1,2,3-triphenol)
structures usually showed a much higher antioxidant activity than
other analogues as a result of the formation of the very stable
semiquinone radicals characterized by extension of the radical
delocalization over anthocyanidin rings B and C, and the two
Páez-Hernández, Rodríguez, & Galán-Vidal, 2009). The most com-
mon anthocyanins are those with the 3-glycoside structure
(Kong, Chia, Goh, Chia, & Brouillard, 2003). Functions of antho-
cyanins in plants include insect attraction for pollination and plant
protection from bacteria and oxidative stress. They have shown a
wide range of pharmacological applications against various stress
conditions and chronic diseases, e.g., inflammation, cognitive
decline, neural dysfunction, capillary fragility and permeability,
⇑
Corresponding author.
E-mail address: Hussein_galaleldeen@agr.asu.edu.eg (H.M. Ali).
http://dx.doi.org/10.1016/j.foodchem.2015.09.003
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

1276
H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
ˇ
hydroxyl oxygen atoms (Azevedo et al., 2010; Castaneda-Ovando
et al., 2009). The resulting radicals can also be stabilized by hydro-
gen bonding with neighboring hydroxyl groups (Foti, Barclay, &
Ingold, 2002; Pereira, Donate, & Galembeck, 1997). It has been
reported that delphinidin (Dp) and Dp-3-glu have a greater DPPH
scavenging activity (Kähkönen & Heinonen, 2003) while cyanidin
showed more antioxidant activity (Kong et al., 2003) than vitamin
E. Glycosylation parameters, i.e., site and number of glycosylation
and sugar type, also affect the antioxidant capacity of anthocyanins
(Zhao et al., 2014). These effects were contradicted in another
study (Wang, Cao, & Prior, 1997). For example, 3-glucosylation
can either increase (Pt and Pg), unaffect (Mv and Cn) or decrease
(Dp and Pn) the antioxidant activity. Type of sugar showed differ-
ent effects on anthocyanin activity; while no significant difference
in activity was observed between cyanidin-3-glycoside with glu-
cose or galactose, cyanidin-3-arabinoside showed significantly less
activity (Kähkönen & Heinonen, 2003). Furthermore, the kind of
target free radical can affect the order of anthocyanidin activity;
cyanidin exhibited a similar activity to malvidin, but less than that
of petunidin, towards the superoxide radical, while cyanidin and
petunidin showed similar activities, but higher than that of mal-
vidin, against the peroxynitrite radical (Rahman, Ichiyanagi,
Komiyama, Hatano, & Konishi, 2006).
2.2. Evaluation of the radical scavenging activity
2.2.1. DPPH radical scavenging activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging
ability of compounds was measured according to Brand-
Williams, Cuvelier, and Berset (1995) with some modifications.
Methanolic DPPH solution was prepared at 0.1 mM (0.004%) con-
centration then stored at ꢀ20 °C. A compound (25
25 l methanol (as a control) was completed to 2.5 ml by DPPH
solution (final concentration 30.0 M). Absorbance (A) was mea-
ll, 3 mM) or
l
l
sured at 517 nm at various intervals until a steady state was
reached; methanolic solution of the compound served as blank.
All experiments were done in duplicates. The inhibitory percentage
of DPPH was calculated according to the following equations:
%DPPH radical scavenging activity
A517ðcontrolÞ ꢀ A517ðExpÞ
¼
ꢁ 100
A517ðcontrolÞ
where A517(Exp) and A517(control) are the absorbance of experi-
ment and control respectively at the steady state condition.
A trolox standard curve of nine concentrations in the linear
range of 1.2–6.0 mM was prepared, and the trolox equivalent
(TEDPPH), defined as mM trolox has the same activity of 1 mM com-
pound, was calculated.
Despite the importance of structure–activity relationship of
anthocyanins not only in determining the structural features
required to enhance the antioxidant activity but also in finding
the source of this enhancement, the SAR of anthocyanins is still
poorly understood (Jhin & Hwang, 2014) and have received fewer
publications than some other classes of flavonoids (Guzmán,
2.2.2. ABTS radical scavenging activity (TEAC method)
The method of Arnao, Cano, and Acosta (2001) with some mod-
ifications was adopted. Stock solutions of 7.4 mM ABTS in water
and 2.6 mM potassium persulfate (K2S2O8) were prepared. Before
use, equal volumes of the two solutions were mixed and allowed
to react in the dark for 12–16 h at room temperature to generate
the ABTS radical cation. The solution was then diluted with metha-
nol until an absorbance of 1.1 at 734 nm (diluted ꢂ30-fold) was
Santiago,
& Sánchez, 2009; Kähkönen & Heinonen, 2003).
Therefore, this study aims to examine the structural features of
anthocyanidins and anthocyanins required to possess high antiox-
idant activity in addition to studying the effect of these features on
the stability of the formed intermediates during the radical scav-
enging process. The examined structural factors included the num-
ber and position of the hydroxyl and methoxy groups, the counter
anion and the glycosylation of the 3-hydroxyl group.
achieved. A compound (20
(control) was diluted to 3.0 ml with the freshly prepared ABTS+ꢀ
solution (final concentration 10.0 M) then the decrease in absor-
lL, 1.5 mM in methanol) or methanol
l
bance was recorded after 6 min in the dark at 734 nm against a
blank of methanolic solution of the compound. All experiments
were done in duplicates. A standard curve of trolox (1.0–6.5 mM)
was prepared. Results are presented as trolox equivalent of antiox-
idant capacity (TEABTS or TEAC value). The % of ABTSꢀ+ scavenging
activity was calculated by the following equation:
2. Materials and methods
2.1. Chemicals and instruments
A734ðcontrolÞꢀA734ðExpÞ
The anthocyanidins used were apigeninidin (Ap), pelargonidin
(Pg), cyanidin (Cn), delphinidin (Dp), peonidin (Pn), petunidin
(Pt), malvidin (Mv) and quercetagetinidin (Qu). The anthocyanins
used were pelargonidin-3-glucoside (Pg-3-glu), cyanidin-3-
% of ABTSꢀþ scavenging activity ¼
ꢁ100
A734ðcontrolÞ
where A734(Exp) and A734(control) are the absorbance of experi-
ment and control respectively at the steady state.
glucoside
peonidin-3-glucoside (Pn-3-glu), petunidin-3-glucoside (Pt-3-
glu), malvidin-3-glucoside (Mv-3-glu) and malvidin-3-
galactocoside (Mv-3-gal). Other chemicals used were
(Cn-3-glu),
delphinidin-3-glucoside
(Dp-3-glu),
2.2.3. Hydroxyl radical scavenging activity
Hydroxyl radical scavenging activity of the compounds was
measured based on the method of Halliwell, Gutteridge, and
Arouma (1987), with a slight modification to match the high
2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azinobis-(3-ethylbenzo
thiazoline-6-sulfonic acid ammonium salt) (ABTS), 2,4,6-
Tripyridyl-s-triazine (TPTZ), hydrogen peroxide, potassium
persulfate, ferrous sulfate, ferric chloride, EDTA, and methanol.
All chemicals were purchased from Sigma–Aldrich or Fluka
Chemical companies. FT-IR was Shimadzu (Affinity-1); scan range
anthocyanin and anthocyanidin activities as follows: 200
deoxyribose solution (5.6 mM), 200 l H2O2 (2.1 mM) and 200
l
l
l
l
l
of the compound (0.05 mM) or oxygen free water (control), were
placed in a test tube. The Fenton reaction was initiated by the addi-
was 400–4000 cmꢀ1
.
1H NMR spectra were recorded on a Bruker
tion of 200
1 mM HCl); all solutions were oxygen free. The reaction mixture
(1 ml) with a final tested compound concentration 10 M was
ll EDTA (0.2 mM) and 200 lL FeSO4 solution (40 lM in
Ultra Shield Plus instrument at 600 MHz at Aramco Company in
Dammam, SA. The chemical shifts are expressed in (ppm)
downfield from tetramethylsilane (TMS) as internal standard.
Deuteriodimethylsulphoxide (DMSO-d6) was used as a solvent.
The prepared compounds were dried by Labconco freeze drier.
UV–Vis spectra were recorded in methanolic solution of the ana-
lyzed compounds on a Thermo Fisher Scientific instrument model
EVO 300LC Evolution 300. The scan covered the range 200–800 nm.
l
heated at 100 °C for 15 min then the reaction was stopped by addi-
tion of 1 ml 10% trichloroacetic acid (TCA). A one ml solution of 2%
thiobarbituric acid (TBA) and 0.04% butylated hydroxyanisole
(BHA) dissolved in NaOH (100 mM) was added. The mixture was
heated at 100 °C for 15 min then cooled and the absorbance (A)
was measured at 532 nm; oxygen free water containing the same

H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
1277
compound concentration served as blank. All experiments were
done in triplicates. The hydroxyl radical scavenging activity was
calculated according to the following equation:
was done by recrystallization from acetic acid to give 57% dark
orange product. UV–Vis (MeOH): 295, 360 and 440 nm. IR (pellet):
ꢀ1
m
O–H 3400 cmꢀ1
,
m
@C–H 3010 cmꢀ1
,
m
C@C 1440, 1530 and
ꢀ
ꢀ
ꢀ
1580 cmꢀ1
,
m
C–O 1190 and 1340 cm
.
1H NMR (DMSO-d6): pro-
ꢀ
A532ðcontrolÞ ꢀ A517ðExpÞ
%OH radical scavenging activity ¼
ꢁ 100
tons on C30 and C50 (7.20 ppm, d, J 9.0 Hz), C2 and C6 (7.78 ppm,
d, J 9.0). C3 (8.82 ppm, d, J 9.0 Hz), C4 (9.37 ppm, d, J 9.0 Hz), ring
A protons m (6.64, 6.80, 8.32 and 8.61 ppm).
A532ðcontrolÞ
where A532(Exp) and A532(control) are the absorbance of experi-
ment and control respectively at the steady state condition.
2.4.3. Synthesis of 40-hydroxy flavylium chlorides
A trolox standard curve in the linear range of 25–800
lM was
A solution of equimolar (8 mmol) of salicylaldehyde and p-
hydroxy acetophenone in distilled EtOH was cooled to 0 °C in con-
ical flask (25 mL). Gaseous HCl (generated by addition of 98%
H2SO4 on solid NaCl) was gently bubbled through the solution
for 90 min. The mixture was kept at 4 °C for 5 days then filtered.
More precipitate was collected after addition of diethyl ether.
The precipitate was washed with diethyl ether and dried by freeze
prepared and the trolox equivalent (TEOH) was calculated.
2.3. Reducing power determination by FRAP assay
The reducing power of the anthocyanidins and anthocyanins
were performed by the ferric reducing ability of plasma (FRAP)
assay as described by Benzie and Strain (1996) with some modifi-
cations. Three stock solutions (300 mM acetate buffer (3.1 g NaOAc
3H2O and 16.0 ml HOAc in liter buffer solution, pH 3.6), 10 mM
TPTZ solution in 40 mM HCl, and 20 mM FeCl3 solution) were pre-
pared. A FRAP working solution was freshly prepared by mixing
25 ml acetate buffer, 2.5 ml TPTZ solution and 2.5 ml FeCl3 solution
drier to give an 81% yield of dark orange proꢀd1uct. UV–Vis (MeOH):
ꢀ1
ꢀ
ꢀ
300 and 455 nm. IR (pellet):
ꢀ
m
m
Oꢀ–1H 3350 cm
,
m
@C–H 3010ꢀc1m
,
ꢀ
m
C@C 1440, 1530 and 1580 cm
,
C–O 1180 and 1340 cm
.
1H
NMR (DMSO-d6): protons on C30 and C50 (7.20 ppm, d, J 9.0 Hz),
C2 and C6 (8.20 ppm, d, J 9.0). C3 (8.61 ppm, d, J 9.0 Hz) C4
(9.27 ppm, d, J 9.0 Hz), ring A protons m (6.84, 8.56 ppm).
then warmed at 37 °C. The studied compounds (150
were diluted to 3.0 ml with the FRAP solution (final concentration
5.0 M). The increase in absorbance was measured at 593 nm after
ll, 0.1 mM)
2.5. Statistical analysis
l
4 min; the blank contained all reagents except using methanol
instead of the assayed compound. All experiments were done in
duplicates. %FRAP was expressed relative to the highest compound
in reducing power according to the following equation:
One-way ANOVA and linear regressions were performed using
SPSS package (version 16). The ANOVA test was used to examine
the significant difference among compounds’ activity. The LSD
method was used to discriminate among means at a 95% confi-
dence level where significance level 60.05 is considered signifi-
cant. Linear regressions were assessed by the correlation
coefficient (R2), standard error of the estimate (SE), the number
of data point (n), the least significant difference (p), and the 5%-
confidence intervals (in parentheses).
A593ðExpÞ
%FRAP ¼
ꢁ 100
A593ðStrongest reductantÞ
where A593(Exp) is the absorbance of the experiment at a steady
state.
A standard curve of trolox was prepared in the linear range of
0.1–1.0 mM. The reducing power is presented as the FRAP value
expressing the trolox equivalent, TEFRAP (mM trolox/mM
compound).
3. Results and discussion
3.1. General
2.4. Synthesis of 40-hydroxy-flavylium products
To examine the antioxidant activity of anthocyanins and their
structure–activity relationship (SAR), a series of 15 anthocyanidins
and anthocyanins (Table 1) along with the synthesized 40-hydroxy
flavylium compounds were examined as radical scavengers against
DPPH, ABTS and hydroxyl radicals, besides their activity as reduc-
tants in the FRAP assay. Two commonly antioxidants known to
have good antioxidant activities namely trolox, (as a monophenol),
and catechol, (as ortho diphenol), were also inspected for
comparison.
2.4.1. Synthesis of 2,40-dihydroxy chalcone
To a solution of salicylaldehyde (8 mmol), ethanol (4 ml) and
aqueous sodium hydroxide (10%, 4.0 ml), p-hydroxy acetophenone
(8 mmol) was added. The solution was heated and refluxed for
approximately 60 min then cooled in an ice-water bath.
Concentrated hydrochloric acid was added until the product began
to precipitate. The product was cooled for 24 h then filtered and
washed with 5 ml of cold water. The crude product was recrystal-
lized from a methanol–water solvent (45% yield). UV–Vis (MeOH):
40-Hydroxy flavylium chloride and ferric chloride were synthe-
sized as outlined in Fig. 1 to examine separately the effects of the
40-OH group on ring B, and 5,7-diOH groups on ring A, on activity;
the three hydroxyl groups present in most common
anthocyanidins.
O–H (3400 cmꢀ1),
C@O
ꢀ
ꢀ
m
260 and 320 nm. IR (pellet):
m
(1680 cmꢀ1),
C@C (1500, 1590 and 1640 cmꢀ1). 1H NMR
ꢀ
m
(DMSO-d6): protons on C30 and C50 (6.64 ppm, d, J 8.1 Hz), C2 and
C6 (7.37 ppm, d, J 8.1 Hz), C4 and C5 (6.36 ppm, m), C3 and C6
(7.17 ppm, m), Ha (6.69 ppm, d, J 9.0 Hz), Hb (7.72 ppm, d, J
3.2. Scavenging DPPH, ABTS and hydroxyl radicals
9.0 Hz), OH (10.2 ppm, s).
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical is
a reactive
2.4.2. Synthesis of 40-hydroxy flavylium ferric chlorides
hydrogen acceptor frequently used in determining the radical scav-
enging activity of various natural and synthetic compounds since it
is a stable colored radical that can be obtained pure and used in
known concentrations (Nishizawa, Kohno, Nishimura, Kitagawa,
& Niwano, 2005). The ABTS assay depends on the oxidation of
the colorless ABTS molecules by oxidizing agents, e.g., potassium
persulfate to the bluish-green radical cation ABTSꢀ+ which can
abstract a hydrogen atom from good hydrogen atom donors to give
the colorless ABTSH+ radical cation (Lien, Ren, Bui, & Wang, 1999).
To a solution of 4.0 mmol 2,40-dihydroxy chalcones in a mini-
mum amount of acetic acid (ꢂ1.0 ml), a concentrated hydrochloric
acid (1.0 ml) was added. The resulting solution was heated in a hot
water bath until a deep red color was formed (ꢂ30 min.) then
cooled at room temperature. A solution of ferric chloride in acetic
acid (16.2 g FeCl3/100 ml glacial acetic acid) was added until no
more precipitate was produced. The precipitate was filtered and
washed with diethyl ether to remove the acetic acid. Purification

1278
H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
Table 1
Structures, maximum absorbance (kmax), colors and common names of the studied anthocyanidin and anthocyanin chlorides.
OH
R6
R3
R3\
OH
HO
O
Cl-
R5\
0
0
Anthocyanidins & anthocyanins
R3
R6
R3
R5
kmax
Color
Apigeninidin (Ap)
Pelargonidin (Pg)
Cyanidin (Cn)
Delphinidin (Dp)
Peonidin (Pn)
H
OH
OH
OH
OH
OH
OH
OH
Oglu
Oglu
Oglu
Oglu
Oglu
Oglu
Ogal
H
H
H
H
H
H
H
OH
H
H
H
H
H
H
H
H
H
OH
H
H
H
OH
H
OH
OMe
H
481
524
538
551
538
549
547
522
514
527
541
526
540
539
540
Orange
Orange-red
Bluish-red
Bluish-red
Bluish-red
Bluish-red
Bluish-red
Orange-red
Orange
Orange-red
Bluish-red
Orange-red
Bluish-red
Bluish-red
Bluish-red
OH
OMe
OMe
OMe
OH
H
OH
Petunidin (Pt) or myrtillidin
Malvidin (Mv)
Quercetagetinidin (Qu)
Callistephin (Pg-3-glu)1
Kuromanin1 (Cn-3-glu)
Myrtillin (Dp-3-glu)
Pn-3-glu
H
H
OH
OH
H
OH
OMe
OMe
OMe
OMe
OMe
OMe
Pt-3-glu
Oenin chloride (Mv-3-glu)
(Mv-3-gal)1
1
Glu and gal stand for glucoside and galactoside respectively.
A hydroxyl radical differs from the other used free radicals in being
one of the most reactive radical formed naturally in biological sys-
tems (Cheng, Ren, Li, Chang, & Chen, 2002). The radical scavenging
activities and the reducing power of anthocyanidins and antho-
cyanins are shown in Table 2. Results are expressed in both, the
percentage activity and trolox equivalent. The final concentrations
of the assayed compounds in each test experiment (30.0, 10.0 and
most of the percentage scale (0–100%) for better differentiation
among the compounds’ activities and accurate correlation with
their structures. Trolox equivalent, on the other hand, is indepen-
dent of antioxidant concentration in the assay solution, and is thus
better used to compare activities towards different radicals or
oxidants.
Results presented in Table 2 show that most of the studied
anthocyanidins and anthocyanins have higher activities than that
of trolox, a strong antioxidant that bears the basic phenolic
10
lM in % DPPH, ABTS and OH assays respectively) were chosen
so that the anthocyanidin and anthocyanin activities varied over
O
O
O
2\
β
6
3
α
1
2
1\
H
3\
4\
5
4
NaOEt
EtOH
6\
OH
OH
OH
OH
5\
Salicylaldehyde
p-Hydroxy acetophenone
2,4\-dihydroxy chalcone
HCl(g)
EtOH, 0oC
1) HCl/HOAc,
2) FeCl3/HOAc
4
5
A
8
6
7
3
2
C
2\
3\
4\
O
O
B
-
Cl-
FeCl4
6\
OH
OH
5\
4\-Hydroxy flavylium ferric chloride
4\-Hydroxy flavylium chloride
Fig. 1. Synthesis of 40-hydroxy flavylium ferric chloride and chloride.

H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
1279
structure of vitamin E. The most active compounds towards all rad-
A
icals were Qu, Dp, Pt and their anthocyanins, with TEDPPH, TEABTS and
TEOH higher than 1.473 (Pt-3-glu), 3.583 (Pt) and 1.698 (Dp-3-glu)
respectively, while the least active compounds against all radicals
were 40-hydroxy flavyliums, Ap, Pg and Pg-3-glu with TEDPPH, TEABTS
and TEOH less than 0.835 (Pg-3-glu), 2.258 (Pg) and 0.819 (Pg-3-glu),
respectively. Compounds with a trolox equivalent <1.0 in the hydro-
xyl radical assay were 40-hydroxy flavyliums, Ap, Pg and Pg-3-glu, in
the DPPH assay were the same compounds plus Pn-3-glu, and in the
ABTS assay were only 40-hydroxy flavyliums. Anthocyanidins and
anthocyanins showed generally more activity, higher trolox equiva-
lent, towards ABTS than DPPH and hydroxyl radicals.
100
80
60
40
20
0
% ABTS (10.0 μM)
% OH (10.0 μM)
% FRAP (5.0 μM)
3.3. Reducing power of anthocyanidins and anthocyanins
The method differs from the previous determinations in that it
does not involve a hydrogen atom transfer reaction but rather
determines the reducing power of the tested compounds; in other
words, it measures the ability of the anthocyanidins and antho-
cyanins to donate an electron rather than a hydrogen atom. The
method depends on the reduction of the colorless ferric 2,4,6-
tripyridyl-s-triazine complex (Fe3+-TPTZ) to the ferrous form
(Fe2+-TPTZ) by abstracting an electron from a reductant. The reduc-
0
20
40
60
80
100
% DPPH
5
4
3
2
1
0
B
TEDPPH
TEABTS
TEOH
ing power was expressed as percentage (%FRAP at 5.0 lM com-
pound) relative to the most active compound, (Qu, 100%) and as
trolox equivalent, TEFRAP (Table 2). Results showed that all the used
anthocyanidins and anthocyanins, except for Ap and monohydroxy
flavyliums, have a good electron donating ability with TEFRAP > 1.0.
TEFRAP values, for example, 5.90, 3.77 and 3.72 for Qu, Pt-3-glu and
Dp-3-glu respectively. Compounds with a catechol or pyrogallol
moiety on ring
A or B gave the highest reducing capacity
(%FRAP > 36.41, TEFRAP > 2.152). These results are consistent with
previously reported results where the TEFRAP of anthocyanins ran-
ged from 0.9 to 5.2, with the highest activities shown for those
with o-diphenols (Jordheim, Aaby, Fossen, Skrede, & Andersen,
2007).
0
1
2
3
4
5
6
TE FRAP
3.4. Structure activity relationship (SAR)
Fig. 2. Linear correlation of %DPPH with %ABTS, %OH and %FRAP (A), and correlation
of TEFRAP with TEDPPH, TEABTS and TEOH (B).
The scavenging activity against DPPH, ABTS and OH radicals and
the reducing power (FRAP assay) presented in Table 2 and Fig. 2
suggest the following SAR features:
Table 2
Radical scavenging activities and reducing power of anthocyanidins and anthocyanins3.
No
Compound
%DPPH (30.0
l
M)2
TEDPPH
%ABTS (10.0
l
M)2
TEABTS
%OH (10.0
l
M)2
TEOH
0.220
%FRAP (5.0
l
M)2
TEFRAP
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Ap
Pg
Cn
Dp
Pn
Pt
Mv
Qu
Pg-3-glu
Cn-3-glu
Dp-3-glu
Pn-3-glu
Pt-3-glu
Mv-3-glu
Mv-3-gal
4-OHFvFeCl4
4-OHFvCl
Catechol
Trolox
10.20( 0.29)a
36.43( 1.55)
66.67( 2.45)b
87.04( 3.69)c
53.49( 2.27)d
83.03( 3.05)c,e
68.64( 2.91)b
0.198
0.708
1.296
1.691
1.039
1.614
1.334
1.543
0.835
1.193
1.625
0.881
1.473
1.245
1.266
0.182
0.138
1.525
1.042
40.12( 1.36)a
1.913
2.258
3.471
3.908
2.402
3.583
2.949
4.189
2.006
2.681
3.891
2.556
4.395
3.306
3.198
0.714
0.736
1.844
1.041
33.33( 1.20)
47.46( 1.28)a
66.1( 2.38)b
86.1( 1.89)
1.00( 0.04)a
0.059
1.085
2.152
2.531
1.524
2.757
1.565
5.904
1.091
2.893
3.717
1.719
3.770
1.873
2.099
0.024
0.036
1.197
0.949
47.36( 1.61)b,c
72.80( 2.99)d,e
81.96( 3.94)f
50.37( 1.71)b,j
75.14( 3.08)d
61.84( 2.54)g
87.86( 4.22)h
42.06( 1.43)a,c
56.23( 1.91)i
81.59( 3.92)f
53.6( 1.82)i,j
92.17( 4.43)h
69.34( 2.84)e,k
67.06( 2.75)g,k
14.98( 0.51)l
15.44( 0.52)l
38.68( 1.31)a
21.83( 0.74)
0.743
1.434
2.175
1.424
2.049
1.459
1.811
0.819
1.671
1.698
1.347
1.823
1.321
1.547
<0.001
<0.001
1.660
1.120
18.37( 0.57)b,h
36.41( 1.39)c
42.87( 1.88)
25.80( 0.80)d
46.69( 2.05)e
26.49( 1.01)d
100.00( 4.38)
18.47( 0.57)b,h
49.00( 2.15)e
62.95( 2.76)f
29.12( 1.11)d,g
63.86( 2.80)f
31.73( 1.21)g
35.54( 1.56)c
0.40( 0.02)a
65.82( 1.45)b,c
82.71( 2.23)
66.78( 2.40)b,d
76.27( 2.06)e
49.49(1.78)a
72.5( 1.60)f
79.41( 2.25)e,f,g
42.99( 1.82)h
61.38( 2.26)i
83.63( 3.55)c,f
45.31( 1.67)h
75.8( 3.22)g,j
64.07( 2.36)b,i
65.17( 2.40)b,i
7.39( 0.21)a
73.22( 1.98)f
61.06( 2.20)g
76.61( 2.07)e
63.05( 2.27)c,g
69.15( 1.52)d
13.56( 0.49)h
11.19( 0.40)h
72.20( 1.95)f
57.63( 2.07)
1
7.12( 0.20)a
0.60( 0.02)a
78.46( 2.88)e,j
53.63( 1.52)d
20.28( 0.63)b
16.06( 0.70)h
1
2
3
Fv stands for flavylium.
Final concentration of the examined compound.
Means ( SD) sharing the same letter in one column are significantly not different at significance level 0.05.

1280
H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
The 40-hydroxy flavylium chloride (Table 2, entry 17) showed
coplanarity of ring B with rings A and C, allowing extension of con-
jugation and electron delocalization leading to extra stability of the
resulting radicals (Balasundram, Sundram, & Samman, 2006; Heim,
Tagliaferro, & Bobilya, 2002).
low scavenging activity towards all radicals (%DPPH 7.12, %ABTS
15.44, %OH 11.19) since the resulted phenoxyl radical can only
be stabilized by a 40-OH radical resonance through the pyrylium
oxygen as presented in Fig. 3. Different counter ions (Clꢀ and FeCl4ꢀ)
showed insignificant effects on the flavylium ion activity in all
tests.
Cyanidin (Cn), which has more hydroxyl groups in 30 position
than Pg, showed much higher scavenging activity (%DPPH 66.7%).
The higher activity of Cn is due to its catechol structure that allows
the well-known stabilization of a semiquinone radical and the for-
mation of a stable quinone product (Ali et al., 2013; Bendary,
In apigeninidin (Ap), the presence of the 5,7-dihydroxyl groups
caused an insignificant difference in activity towards DPPH (%DPPH
10.20) but a significant increase in %ABTS (40.12) and %OH (33.33).
Despite the more hydroxyl groups in Ap, its radical, upon dehydro-
genation, can only give 5- or 7-OH radical resonance through the
pyrylium oxygen similar to that of the 40-hydroxyl group as
depicted in Fig. 3. Apigeninidin showed different behavior towards
different radicals with no activity towards hydroxyl and nitric
oxide radicals, but a good activity towards lipids and ascorbyl rad-
icals (Boveris et al., 2001). Chrysin has a similar structure to that of
apigeninidin but with no 40-OH group. Both anthocyanidins gave
similar scavenging activities towards the ABTS radical cation
(Rice-Evans, Miller, & Paganga, 1996) indicating similar stabiliza-
tion effects of 40-OH radical resonance and 5- or 7-OH radical
resonance.
Pelargonidin (Pg) has a similar structure to Ap but with an extra
hydroxyl group in position 3; it gave significantly higher activities
against all radicals. The higher activity can be attributed to the
3-OH radical resonance between 3-OH and 5-, 7- or 40-OH groups,
and the formation of a stable diketonic product as illustrated in
Fig. 3, which pushes the hydrogen transfer reaction forward.
Radicals formed on 5-, 7- or 40-hydroxyl groups can also be stabi-
lized by the same resonance in the opposite direction. This reso-
nance effect is similar to that reported for the catechol structure
where stable semiquinone radicals are formed leading to stable
ˇ
Francis, Ali, Sarwat, & El Hady, 2013; Castaneda-Ovando et al.,
2009) as illustrated in Fig. 3. Petunidin (Pt) showed more scaveng-
ing activity against all radicals (e.g., %DPPH 83.03) than that of Cn
since it bears a catechol structure but one more OMe group. It can
also be observed that the strongest reductants in the FRAP assay
were Dp, Pt and Cn, and their anthocyanins plus Qu, indicating that
a catechol moiety enhances not only radical scavenging activity
but also the electron donating ability of anthocyanidins and antho-
cyanins as mentioned above.
Delphinidin (Dp) has more hydroxyl groups in the 50 position
than Cn (pyrogallol structure) on ring B, which increased scaveng-
ing activity towards all radicals (e.g., %DPPH 87.04). Quercetage-
tinidin (Qu) has a pyrogallol moiety on ring A and a catechol
moiety on ring B. It gave also higher activity against the three rad-
icals (e.g., %DPPH 79.41) than that of Cn.
According to the previous SAR analysis, peonidin (Pn) should
give less activity (%DPPH 53.49) than Cn (%DPPH 66.67) since it
has the same structure with methylation of the 30-hydroxyl group,
and thus lacks the catechol structure. On the other hand, it has a
higher activity than that of pelargonidin because of the extra
30-OH group; similar results were obtained by Kähkönen
&
Heinonen, 2003. As peonidin, malvidin (Mv) lacks a catechol struc-
ture but has more 50-OMe groups; it gave a higher activity (%DPPH
68.64). The electronic effect of substituents has a large influence on
both the activation energy and O–H bond dissociation energy of
phenols (Foti, Daquino, Mackie, DiLabio, & Ingold, 2008). It was
reported previously that methoxy and hydroxyl groups stabilize
ˇ
diketones (Ali & Ali, 2015; Castaneda-Ovando et al., 2009). The
presence of the 3-OH group conjugated with a 2–3 double bond
was previously reported to enhance the antioxidant activity in
other classes of flavonoids; this structural feature permits
O
O
O
O
4\-hydroxyl radical resonance
OH
O
O
OH
O
HO
HO
O
O
O
O
O
5-hydroxyl radical resonance
7-hydroxyl radical resonance
H
O
O
O
O
O
O
O
- H
- H
OH
O
O
HO
HO
Semiquinone radical resonance
3-hydroxyl radical resonance
Fig. 3. Stabilization resonance of various anthocyanidin radicals.

H.M. Ali et al. / Food Chemistry 194 (2016) 1275–1282
1281
100
80
60
40
20
donating ability of anthocyanidins and anthocyanins, since linear
correlations can be drawn between %DPPH and other assays as pre-
sented in Fig. 2A and regressions 1–3. Correlations show that the
slope of the three regressions are very close (R2 = 0.75–0.81) indi-
cating not only that factors affecting scavenging the three radicals
and reducing power are similar, but also their contributions have
similar weights. High correlations (R2 > 0.9) were recently reported
between any two of DPPH, ABTS and FRAP results (Jiméneza et al.,
2015). Besides, Fig. 2B shows that anthocyanidins and antho-
cyanins were more active towards an ABTS radical cation than
OH followed by DPPH radicals.
% DPPH
% ABTS
% OH
% FRAP
%ABTS ¼ 17:65ðꢃ5:57Þ þ 0:77ðꢃ0:09Þ%DPPH
ðReg:1Þ
ðReg:2Þ
ðReg:3Þ
R2 0.842, SE 8.393, n 16 (entries 1–16), F 74.40, p 0.000.
%OH ¼ 18:94ðꢃ4:01Þ þ 0:75ðꢃ0:06Þ%DPPH
0
R2 0.908, SE 6.041, n 16 (entries 1–16), F 137.70, p 0.000
%FRAP ¼ ꢀ9:98ðꢃ10:48Þ þ 0:81ðꢃ0:17Þ%DPPH
R2 0.623, SE 15.787, n 16 (entries 1–16), F 23.21, p 0.000.
Pg
Pn
Mv
Cn
Pt
Dp
Anthocyanidin
100
Reg. (3) was improved when Qu, of exceptionally high reducing
power, was eliminated to give Reg. (4).
80
60
40
20
0
%FRAP ¼ ꢀ6:04ðꢃ6:14Þ þ 0:68ðꢃ0:10Þ%DPPH
R2 0.782, SE 9.176, n 15, F 46.55, p 0.000.
ðReg:4Þ
The anthocyanins (Table 2, entries 9–15) gave a similar trend to
those of their respective anthocyanidins as presented in Fig. 4.
Glycosylation of anthocyanidins may cause an increase or decrease
in their activity. In the DPPH test for example, Cn, Pn, and Pt gave
significantly higher activities while Pg gave a significantly lower
activity than their respective anthocyanins; others, e.g., Dp and
Mv showed insignificant difference. The diverse results could be
attributed to the contradicted effects of the sugar unit. Jing et al.
(2014) showed that the higher activity of 3-glucoside anthocyanins
than their aglycons could be due to the electron donating effect of
the 3-bulky sugar group. On the other hand, Kähkönen and
Heinonen (2003) reported that diglucosidation of anthocyanidins
at the 3 and 5 positions lowered the activity which is consistent
with prohibition of stabilization by both 3 and 5-radical reso-
nances outlined in Fig. 3. Zhao et al. (2014) have also demonstrated
in their recent review that the glycosylation of anthocyanidins usu-
ally lessen their antioxidant activity but may also enhance activity
depending on the anthocyanidin type and the experimental
method.
Pg-3-glu
Pn-3-glu
Mv-3-glu
Cn-3-glu
Pt-3-glu
Dp-3-glu
Anthocyanin
Fig. 4. Radical scavenging and reducing activities of some anthocyanidins and their
respective anthocyanins.
the resulted radicals of phenolic compounds, leading to an increase
in their antioxidant activity with a greater effect shown by the
methoxy group than hydroxyl substituent (Ali
& Ali, 2015;
Balasundram et al., 2006; Heim et al., 2002). Therefore, comparing
the activity of compounds lacking the catechol structure, we find
the activity against the three radicals (DPPH, ABTS and OH)
decreases in the order of Mv > Pn > Pg > Ap > 4-OH-flavylium
respective to the presence of 6, 5, 4, 3 and 1 hydroxyl and/or meth-
oxy groups; the difference is significant in most cases. The same
order of activity (Mv > Pn > Pg) was previously found experimen-
tally in the ORAC test (Wang et al., 1997) and predicted theoreti-
cally based on the calculation of O–H bond dissociation energies
(Guzmán et al., 2009). In addition, the same order was found in
the reducing power measured by the FRAP assay (Table 2) suggest-
ing similar factors affecting both the reducing power and the rad-
ical scavenging activities of anthocyanidins and anthocyanins. It
was observed previously that the reducing power increases with
the number of hydroxyl and methoxy groups on ring B (Azevedo
et al., 2010; Balasundram et al., 2006). It can also be observed that
a compound which lacks a catechol structure, e.g., Mv with four
hydroxyl and methoxy groups may have scavenging activities
similar or even better (e.g., DPPH and OH assays) than that of a
compound possessing a catechol structure, e.g., Cn with less stabi-
lizing substituents.
4. Conclusion
It can be concluded from the present study that the widely
distributed anthocyanidins and anthocyanins proved to be strong
radical scavengers against a variety of free radicals, i.e., DPPH, ABTS
and OH radicals where most of the anthocyanidins and antho-
cyanins showed more scavenging activity than that of the well-
known strong antioxidants trolox and catechol. Anthocyanidins
and anthocyanins scavenging activities were sensitive towards
the number and position of the hydroxyl and methoxy groups.
Compounds with a catechol structure in either ring A or B were
generally better hydrogen atom donors because of the stabilization
of the resulted radicals and the formation of stable quinone-like
products; besides, they were much stronger electron donors. The
presence of hydroxyl groups in positions 5, 7 and 40 only has little
effect on both hydrogen atom and electron donation activities,
while the presence of a 3-OH group improved both of these activ-
ities. The importance of 3-OH group in hydrogen transfer reactions
suggests a 3-radical resonance through 5 or 7-OH group forming an
anthocyanin semiquinone radicals ending with a stable diketone,
similar to that observed for the catechol structure. On the other
The discussed SAR features can be applied on the results of
scavenging the three studied radicals as well as the electron

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