Polyphenolics from various extracts/fractions of red onion (Allium cepa) peel

with potent antioxidant and antimutagenic activities

Brahma N. Singh
a
, B.R. Singh
b
, R.L. Singh
c
, D. Prakash
a
, D.P. Singh
d
, B.K. Sarma
d
, G. Upadhyay
a
,
H.B. Singh
d,
*
a
Nutraceutical Chemistry, National Botanical Research Institute, Lucknow 226 001, India
b
Agricultural Microbiology, Institute of Agricultural Sciences, Aligarh Muslim University, Aligarh 202 001, India
c
Department of Biochemistry, Dr. R.M.L. Avadh University, Faizabad 224 206, India
d
Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221 005, India
a r t i c l e i n f o
Article history:
Received 30 September 2008
Accepted 3 February 2009
Keywords:
Allium cepa
HPLC
Antioxidant activity
Protein fragmentation
Ames test
DNA damage
a b s t r a c t
In order to determine antioxidant activity, the ve extracts/fractions of red onion peel were studied for
their total content of phenolics (TPC), avonoids (TFC), antioxidant activity (AOA), free radical scavenging
activity (FRSA), assayed by DPPH radical in the terms of anti-radical power (ARP) and reducing power
(RP), expressed as ascorbic acid equivalents (ASE)/ml. High TPC (384.7 5.0 mg GAE/g), TFC
(165.2 3.2 mg QE/g), AOA (97.4 7.6%), ARP (75.3 4.5) and RP (1.6 0.3 ASE/ml) were found for the
ethyl acetate (EA) fraction. EA fraction had markedly higher antioxidant capacity than butylated hydroxy-
toluene (BHT) in preventive or scavenging capacities against FeCl
3
-induced lipid peroxidation, protein
fragmentation, hydroxyl (site-specic and non-site-specic), superoxide anion and nitric oxide radicals.
EA fraction also showed dose dependent antimutagenic activity by following the inhibition of tobacco-
induced mutagenicity in Salmonella typhimurium strains (TA102) and hydroxyl radical-induced nicking
in plasmid pUC18 DNA. HPLC and MS/MS analysis showed the presence of ferulic, gallic, protocatechuic
acids, quercetin and kaempferol. The large amount of polyphenols contained in EA fraction may cause its
strong antioxidant and antimutagenic properties. This information shows that EA fraction of red onion
peel can be used as natural antioxidant in nutraceutical preparations.
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Reactive oxygen species (ROS) are continuously produced in
biological system as products or by-products of plethora of enzy-
matic reactions and also produced by exogenous sources including
tobacco, smoke, radiations, auto-exhaust and pesticides. These ROS
are able to oxidize cellular bio-molecules like nucleic acids, pro-
teins, lipids and carbohydrates (Ardestani and Yazdanparast,
2007; Bendich, 1996; Halliwell, 1995). Their damage plays a signif-
icant pathological role in aging, cancer, cardiovascular, inamma-
tory and neurodegenerative diseases (Borek, 1997; Sun, 1990;
Wiseman and Halliwell, 1996). It has been reported that the diets
rich in vegetables and fruits provide a great amount of antioxidant
phytochemicals, such as polyphenolics, carotenoids, terpenoids,
avonoids and vitamins E and C, glutathione, and vegetable pig-
ments, which offer protection against cellular damage due to their
ability to quench oxygen-derived free radicals by donating elec-
tron, chelate to redox-active metals and inhibit lipooxygenases
(Dimitrios, 2006; Patricia et al., 2005).
Onion (Allium cepa) is a versatile vegetable that is consumed
fresh as well as in the form of processed products. More recently,
there has been renewed attention given to the antioxidant content
of onions because many epidemiological studies suggested that
regular consumption of onions in food is associated with a reduced
risk of neurodegenerative disorders, many forms of cancer, cataract
formation, ulcer development, reduction in symptoms associated
with osteoporosis (NOA), prevention of vascular and heart diseases
by inhibition of lipid peroxidation (LPO) and lowering of low den-
sity lipoprotein (LDL) cholesterol levels (Kaneko and Baba, 1999;
Kawaii et al., 1999; Sanderson et al., 1999; Shutenko et al., 1999).
Onion is one of the major sources of various biologically active
phytomolecules e.g. phenolic acids, avonoids, cepaenes, thiosulf-
inates and anthocyanins (Goldman et al., 1996). The major avo-
noids found in dry peel of onion that has been considered
usually as waste, contain large amounts of quercetin, quercetin
glycoside and their oxidative product which are effective antioxi-
dants against the lethal effect of oxidative stress (Glsen et al.,
2007; Prakash et al., 2007c). They are also reported to have liver
protective effect, immune enhancement potential and anti-infec-
tion, anti-stress, anti-cancer and other pharmacological properties
(Balasenthil et al., 1999; Valko et al., 2007).
0278-6915/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.02.004
* Corresponding author. Tel.: +91 542 2307116; fax: +91 542 2368993.
E-mail address: hbs1@rediffmail.com (H.B. Singh).
Food and Chemical Toxicology 47 (2009) 11611167
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Previously, we found that outer layer of red onion had the high-
est antioxidants and antioxidant activities than the purple, white
and green varieties of onion, as determined by in vitro antioxidant
and free radical scavenging activities (Prakash et al., 2007a). It is
interesting that onion peels have been used all over the world as
colorant, avor agent, and various types of food. Available informa-
tion on their antioxidant activity and antimutagenic potential is
scanty. In the current study, we optimized the antioxidant extract-
ing procedure, based on their contents of total phenolics (TPC),
avonoids (TFC), antioxidant activity (AOA), free radical scaveng-
ing activities (FRSA), and reducing power (RP) using standard
in vitro antioxidant assays. Specic phenolics composition using
high performance liquid chromatography (HPLC) and mass spec-
troscopy (MS)/MS in the various extracts/fractions was also per-
formed. Extracts/fractions examined were prepared using
successive solvents of varying polarity and by partitioning the
methanol fraction with diethyl ether, ethyl acetate and n-butanol.
The peel extract/fraction, with high TPC, TFC, AOA, FRSA, RP and
specic phenolics content, that we obtained was further evaluated
by using different in vitro antioxidant models for their antioxidant
activity, including preventive or scavenging capacity against hy-
droxyl, superoxide anion and nitric oxide radicals, as well as inhib-
iting FeCl
3
-induced LPO. Antimutagenic potential of promising
extract/fraction was also studied on Salmonella typhimurium TA-
102 tester strain and pUC18 plasmid DNA using Ames and DNA
damage tests, respectively. The objectives of this study were to
establish an efcient antioxidant extracting procedure and to ex-
plore the possibility of developing a nutraceutical agent rich in nat-
ural antioxidants from the red onion peel.
2. Materials and methods
2.1. Chemicals and materials
Nitro blue tetrazolium (NBT), gallic acid, ferulic acid, ellagic acid, protocatechuic
acid, quercetin, kaempferol, pUC18 DNA, 1,1-diphenyl-2-picrylhydrazyl (DPPH),
thiobarbituric acid (TBA), phenazine methosulphate (PMS), nicotinamide adenine
dinucleotide (NADH), sodium nitroprusside and 2,4-dinitrophenylhydrazine
(DNPH) were purchased from SigmaAldrich, St. Louis, USA. Linoleic acid, b-caro-
tene, butylated hydroxytoluene (BHT), and 2-deoxy-D-ribose sugar were purchased
from Acros, Organics, Geel, Belgium. Tween 40, Folin Ciocalteaus reagent, human
serum albumin (HAS), and other analytical grade chemicals were obtained from
E. Merck, Mumbai, India. Red onion (variety N-53) was collected from the exper-
imental eld of Department of Horticulture, Institute of Agricultural Sciences, Ban-
aras Hindu University, Varanasi, India. Dry peel of red onion was separated and
further dried at 40 C for 8 h, powdered (40-mesh) and stored in polythene bags
at 4 C.
2.2. Extraction procedure
Thousand grams of dried powder of onion peel was put in a Soxhlet apparatus
and extracted successively with toluene, dichloromethane, ethanol of increasing
polarity until decolouration. The successive extracts were evaporated at 40 C in a
vacuum rotary evaporator (Eyela NN series, Tokyo, Japan) to yield 3.17 g of toluene
extract, 2.61 g of dichloromethane (DCM) extract and 50.38 g of ethanol extract.
Fifty grams of the ethanol dry extract was dissolved in 1.5 l water (Wa), ltered
and partitioned with diethyl ether (DEE), ethyl acetate (EA) and n-butanol (Bu). Or-
ganic layers of each of the three solvents were dried with anhydrous sodium sul-
phate, ltered and evaporated under vacuum up to dryness to yield 1.14 g of DEE
fraction, 4.79 g of EA fraction, 5.16 g of Bu fraction and 3.13 g of the remaining
Wa fraction (Anagnostopoulou et al., 2006; Mellidis et al., 1993).
2.3. Biochemical analysis
Total phenolic content (TPC) was estimated as described by the method of Rag-
azzi and Veronese (1973) and expressed as mg gallic acid equivalent (GAE)/g dry ex-
tract. Total avonoid content (TFC) was estimated as described by Oyaizu (1986) and
expressed as mg quercetin equivalent (QE)/g extract. Antioxidant activity (AOA) was
performed by autoxidation of b-carotene and linoleic acid coupled reaction method
Emmons and Peterson (1999) and expressed as percentage inhibition, relative to
control. Free radical scavenging activity (FRSA) was measured by using DPPHradical
solution (6 10
5
M in HPLC grade methanol) according to Yen and Duh (1994) and
expressed in the terms of antiradical power (ARP) described by Kroyer (2004).
Reducing power (RP) was determined by ferric reducing antioxidant power assay
and expressed as ascorbic acid equivalents (ASE) per milligram (Apati et al., 2003).
The ASE value is inversely proportional to RP. Inhibition of LPO was determined
by using rat liver homogenate as a lipid rich source by the method of Ohkowa
et al. (1979). Protein fragmentation assay was carried out according to the method
of Ardestani and Yazdanparast (2007) with some modication and results were ex-
pressed as percentage inhibition of protein carbonyl (PCO) formation. Non-site-spe-
cic and site-specic hydroxyl radical-mediated 2-deoxy-D-ribose degradation was
performed as described by Halliwell et al. (1987). Superoxide anion radical and nitric
oxide radical scavenging activities were examined by the methods of Nishikimi et al.
(1972) and Chi et al. (2001), respectively.
2.4. HPLC analysis
All the ve extracts/fractions of onion peel were acid hydrolysed with 1.2 N HCl
by reuxing on a water bath for 2 h. The hydrolysates were dissolved in HPLC grade
methanol (1.0 mg/ml), ltered through sterile 0.22 lm millipore lter and sub-
jected to qualitative and quantitative analysis by using Shimadzu LC-10A (Kyoto, Ja-
pan) HPLC instrument. The instrument equipped with a dual-pump LC-10AT binary
system (Shimadzu, Kyoto, Japan), a UV detector SPD-10A (Shimadzu, Kyoto, Japan),
and a Phenomenex Luna RP, C 18 column (4.6 250 mm). Data were integrated by
Shimadzu Class VP series software (Shimadzu, Kyoto, Japan). Separation was
achieved with an acetonitrile/water containing 1% acetic acid linear gradient pro-
gram, started with 18% acetonitrile, changing to 32% in 15 min and nally to 50%
in 40 min. Results were obtained by comparison of peak areas (k
max
254 nm) of
the plant samples (lg/g dry weight) with that of standards (Prakash et al., 2007a).
2.5. Qualitative analysis by MS/MS
An API 2000 triple quadrupole mass spectrometer (Applied Biosystems, Ontario,
Canada) was used for identication of phenolic compounds. Analysis were per-
formed on a Turbo ions spray source in negative mode by using settings, nebuliser
gas (N
2
) 16, curtain gas (N
2
) 12, collision gas (N
2
) 12 (arbitrary units), focusing po-
tential 400 V, entrance potential 10, declustering potential (DP) 2560 and col-
lision energy (CE) 1535. Full scan acquisition was performed by scanning from m/z
1001000 u at a cycle time of MS/MS product ions were produced by collision-asso-
ciated dissociation (CAD) of the selected precursor ions in collision cell. In all the
experiments, quadrupole (Q
1
) was operated at unit resolution. Product ion scan of
selected molecules were carried out in order to conrm the structure of the com-
pounds (Prakash et al., 2007c).
2.6. Antimutagenic activity
2.6.1. Ames test
To examine the antimutagenic activity of plant extract against the mutagenicity
of tobacco extract using TA102 tester strain of S. typhimurium by standard plate
incorporation test was carried out according to the slightly modied method of
Maron and Ames (1983). For preparation of tobacco extract, 50 g leaves was cut into
small pieces and boiled in 200 ml of distilled water for 1 h. It was evaporated to dry-
ness (Santhosh et al., 2005). 1.0 ml of fresh bacterial culture (12 10
8
cells/ml)
was mixed with 2.0 ml of top agar containing 0.045 mM histidine and biotin, vari-
ous concentrations of plant extract and tobacco extract (50 mg/plate). Further, it
was poured onto minimal glucose-agar plate and incubated for 48 h at 37 C. After
expiry of the period, numbers of revertant colonies were counted after incubation of
the plates. Positive and negative controls were also included in each assay. Tobacco
extract was used as a diagnostic mutagen in positive control plates. Negative con-
trols were prepared with equivalent amount of propylene glycol instead of tobacco
extract and plant extract, which is required to establish the number of colonies that
arise spontaneously for S. typhimurium. The mutagenicity of tobacco extract in the
absence of plant sample was dened as 100% or 0% inhibition. The calculation of
percent inhibition was done according to the formula given by Ong et al. (1986),
% inhibition (1-T/M)100, where T is number of revertants per plate in presence
of mutagen and plant sample and M is number of revertants per plate in positive
control (tobacco extract).
2.6.2. DNA damage
To evaluate the antimutagenic potential, plasmid DNA nicking assay was per-
formed using supercoiled pUC18 plasmid DNA according to the method of Lee
et al. (2002) with some modication. Plant extract (10 ll) of different concentra-
tions (210 lg/ml) and puried pUC18 DNA (0.25 lg) were incubated for 10 min
at room temperature followed by the addition of 10 ll Fentons reagent (30 mM
H
2
O
2
, 50 lM ascorbic acid and 80 lM FeCl
3
). The reaction mixture was incubated
for 45 min at 37 C and analyzed on 0.9% agarose gel followed by ethidium bromide
staining.
2.7. Statistical analysis
Results presented in tables and graphs were reported as mean standard devi-
ation (SD) of at least three replicates of the same sample. Data were subjected to
1162 B.N. Singh et al. / Food and Chemical Toxicology 47 (2009) 11611167
one-way analysis of variance (ANOVA) and the least signicant difference (LSD) at
P < 0.01 was calculated by post-hoc comparison test (SPSS 11.5) to determine signif-
icant differences between the various extracts/fractions as well different doses of
promising extract/fraction of red onion peel.
3. Results and discussion
3.1. Antioxidant activity
All the ve extracts/fractions of red onion peel were studied for
their total contents of phenolics (TPC), avonoids (TFC) and antiox-
idant activity (AOA) (Table 1). TPC showed wide variation from
384.7 5.0 (EA fraction) to 23.1 0.9 mg GAE/g extract (DCM ex-
tract) and TFC from 165.2 3.2 (EA fraction) to 1.3 0.21 mg QE/g
extract (DCM extract). AOA varied from 14 1.7 (DCM extract) to
97.4 7.6% (EA fraction) as measured by auto-oxidation of b-
carotene and linoleic acid coupled reaction. TPC, TFC and AOA of
extracts/fractions followed similar trend and descended in the fol-
lowing order: EA fraction > Bu fraction > DEE fraction > Wa frac-
tion > DCM extract. As it can be seen, the methanolic fractions
have the highest TPC, TFC and AOA. Ethyl acetate seems to be the
solvent that concentrates best antioxidant substances (i.e. poly-
phenols) of intermediate polarity. This is in accordance with nd-
ings by Anagnostopoulou et al. (2006), Chung et al. (1999) and
Parejo et al. (2002).
Free radical scavenging activity (FRSA) is very important due to
the deleterious role of free radicals in food and in biological sys-
tems (Barrett, 1991). Since phenols and avonoids are known to
be responsible for FRSA (Prakash et al., 2007c), the extracts/frac-
tions of red onion were also studied for their FRSA assayed by
DPPH radical (Table 1). FRSA expressed in terms of anti-radical
power (ARP) ranged from 1.2 0.3 in DCM extract to 75.3 4.5 in
EA fraction and varied in the same order as has been observed
for TPC, TFC and AOA. The ARP of EA fraction (75.3 4.5) was found
to be better than that of standard BHT (30.9 2.1) used as a known
free radical scavenger, suggesting that EA fraction has contained
maximum natural antioxidants from red onion peel. To measure
reducing power (RP) of extracts/fractions, we have investigated
the Fe
3+
Fe
2+
reducing potential using potassium ferricyanide
method and results were expressed in terms of ascorbic acid equiv-
alents (ASE) per millilitre (Table 1). The ASE value is inversely pro-
portional to reducing power (Apati et al., 2003). Reducing ability
was found to be maximum in EA fraction (1.6 0.3 ASE/ml) and
minimum in DCM extract (7.9 0.8 ASE/ml). The EA and Bu frac-
tions have better Fe
3+
Fe
2+
transformation ability than that of
BHT (3.1 ASE/ml), a reference standard (Table 1).
All the extracts/fractions were further examined for their spe-
cic phenolics composition by the HPLC to evaluate the presence
of phenolic acids and avonoids (Table 2). The quantity of phenolic
compounds ranged from 4.1 1.1 to 74.9 4.8 lg/g gallic acid,
2.0 0.5 to 19.6 2.3 lg/g ferulic acid, 5.07 1.0 to 53.3 2.0 lg/
g protocatechuic acid, 4.7 0.3 to 3916.9 29.7 lg/g quercetin
and 1.8 0.1 to 231.4 6.5 lg/g kaempferol. The EA fraction was
found to be the richest source of polyphenolics such as ferulic acid
(19.6 2.3 lg/g), protocatechuic acid (53.3 2.0 lg/g), quercetin
(3916.9 29.7 lg/g) and kaempferol (231.4 6.5 lg/g) in compar-
ison to other extracts/fractions. Highest quantity of gallic acid
(74.9 4.8 lg/g) was detected in the Bu fraction. However the
presence of ferulic acid and protocatechuic acid, quercetin and
kaempferol could not be traced in Wa fraction. Similarly, gallic acid
Table 1
Antioxidants and antioxidant activities of red onion peel.
Sample TPC
a
TFC
b
AOA
c
ARP
d
RP
e
DCM extract 23.1 0.9e 1.3 0.4de 14.0 1.7ef 1.2 0.3def 7.9 0.8ab
Methanolic extract
DEE fraction 47.3 1.5c 9.1 1.23c 24.2 2.6def 4.9 0.6def 5.0 0.4c
EA fraction 384.7 5.0a 165.2 3.2a 97.4 7.6ab 75.3 4.5a 1.6 0.3def
Bu fraction 102.1 1.8b 57.8 2.1b 69.7 5.7c 13.4 0.8c 2.9 0.4def
Wa fraction 27.3 1.7d 1.5 0.21de 31.5 3.4d 1.8 0.3def 7.3 0.9ab
BHT 91.9 6.4ab 30.9 2.1b 3.1 0.5def
P ANOVA
*** *** *** *** ***
Values are the mean SD (n = 3). Data were analysed by ANOVA (
***
P < 0.001) and within each column different letters indicate statistically different values according to post-
hoc comparison (LSD-test) at P < 0.01.
a
TPC, total phenolics content mg gallic acid equivalents (GAE)/g extract.
b
TFC, total avonoids content mg quercetin equivalents (QE)/g extract.
c
AOA, antioxidant activity (%).
d
ARP, antiradical power.
e
RP, reducing power expressed as ascorbic acid equivalents (ASE)/ml.
Table 2
Specic phenolic composition in peel of red onion.
Sample Phenolic content
a
(lg/g dry weight)
Gallic acid Ferulic acid Protocatechuic acid Quercetin Kaempferol
DCM extract 2.0 0.5cd 4.7 0.3cd 1.8 0.1d
Methanolic extract
DEE fraction 17.4 1.8bc 4.7 0.7cd 5.07 1.0c 35.7 7.5cd 13.1 0.4c
EA Fraction 22.5 2.8bc 19.6 2.3a 53.3 2.0a 3916.9 29.7a 231.4 6.5a
Bu fraction 74.9 4.8a 11.1 0.9b 8.6 0.8b 1358.1 12.0b 47.7 3.7b
Wa fraction 4.1 1.1cd
P ANOVA
*** *** *** *** ***
a
Values are the mean SD (n = 3). Data were analysed by ANOVA (
***
P < 0.001) and within each column different letters indicate statistically different values according to
post-hoc comparison (LSD-test) at P < 0.01.
B.N. Singh et al. / Food and Chemical Toxicology 47 (2009) 11611167 1163
and protocatechuic acid were not found in DEE fraction. Our results
suggested that EA fraction of red onion peel can be utilised for the
isolation of phenols and avonoids that have been reported to pro-
vide benecial effects in human health (Bingham et al., 2003).
The identication of specic polyphenols in all the extracts/
fractions of onion peel was further substantiated by MS/MS analy-
sis (Table 3), that showed deprotonated molecules [M-H]

. Loss of
CO
2
was observed for ferulic acid, gallic acid and protocatechuic
acid, giving the ion [M-H-44]

as a characteristic ion. Ferulic acid

also showed the loss of CH
3
group, providing [M-15]

anion rad-
ical at m/z 178 in the product ion scans. Finally, the aglycones such
as quercetin and kaempferol gave RetroDielsAlder fragmenta-
tion to give an ion m/z 151; in case of kaempferol loss of neutral
water molecule gave an ion at m/z 133 also (Sanchez-Rabaneda
et al., 2003).
In the above investigation, EA fraction has shown potent activ-
ity as well as higher in contents of antioxidant phytomolecules as
compared to all the other extracts/fractions. Hence, it is selected
for further antioxidant screening. Peroxidation of lipid may lead
to the formation of several toxic by-products such as malondialde-
hyde (MDA) and 4-hydroxynonenal which can attack on DNA,
inducing mutagenecity and carcinogenicity (Patricia et al., 2005;
2007c; Zwart et al., 1999). Inhibition of LPO by any external agent
is often used to evaluate its antioxidant activity. The antioxidant
activity of EA fraction was examined by inhibition of LPO induced
by Fe
3+
-dependent hydroxyl radicals. Additions of EA fraction at
concentrations of 100, 200, 300, 400 and 500 lg/ml have produced
Table 3
Phenolic compounds identied by MS/MS analysis.
Phenols Ion full scan MS MS/MS Approach
[M-H]

Fragments Product Ion scan

Gallic acid 169 125 169
Ferulic acid 193 178, 149 193
Protocatchuic acid 153 109 153
Quercetin 301 151 301
Kaempferol 285 133, 151 285
0
10
20
30
40
50
60
70
80
90
100
100 200 300 400 500
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
e
d
c
b
a
a
b
c
d
e
0
10
20
30
40
50
60
70
80
90
200 400 600 800 1000
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
ab
ab
c
d
e e
d
c
b
a
0
20
40
60
80
100
120
100 200 300 400 500
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
ab
ab
c
d
e
b a
c
d
e
0
10
20
30
40
50
60
70
80
90
100
100 200 300 400 500
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
ab
abc
bc
d
e
ab
ab
c
d
e
0
20
40
60
80
100
120
100 200 300 400 500
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
e
d
d
e
bc
c
abc
ab
ab
ab
0
20
40
60
80
100
120
100 200 300 400 500
Concentration (g/ml)
%

i
n
h
i
b
i
t
i
o
n
EA fraction BHT
a
b
cd
cd
e
a
b
c
d
e
a
c d
e f
b
Fig. 1. Inhibitory effects of EA fraction and BHT on: (a) lipid peroxidation using rat liver homogenate as a lipid rich source; (b) hydroxyl radical-induced protein carbonyl
(PCO) formation; (c) site-specic hydroxyl radical-mediated deoxyribose degradation; (d) non-site-specic hydroxyl radical-mediated deoxyribose degradation; (e)
superoxide anion radical; and (f) nitric oxide radical. Values are the mean SD (n = 3). Data were analysed by ANOVA (
***
P < 0.001) and within each column different letters
indicate statistically different values according to post-hoc comparison (LSD-test) at P < 0.01.
1164 B.N. Singh et al. / Food and Chemical Toxicology 47 (2009) 11611167
15.32 0.72%, 33.41 1.79%, 52.71 3.0%, 79.41 4.21% and
87.52 3.22% inhibition of hydroxyl radical-mediated LPO, respec-
tively (Fig. 1a). On the other hand, at the same concentrations,
standard BHT exhibited 14.25 0.95%, 29.23 1.47%,
38.84 2.06%, 54.79 2.09% and 71.17 2.97% inhibition, respec-
tively. To further measure the scavenging effect of EA fraction on
Fe
3+
-dependent hydroxyl radical, we initially measured the oxy
radical scavenging activity of EA fraction using protein fragmenta-
tion assay, which measures the degree of protein oxidation in the
terms of inhibition of protein carbonyl (PCO) formation. EA fraction
strongly inhibited Fe
3+
-dependent protein oxidation by
9.75 0.71%, 24.16 1.48%, 49.29 3.49%, 74.51 4.41% and
87.33 4.07% in a concentration-dependent manner of 100
500 lg extract/ml, respectively which was higher than standard
BHT (9.25 1.2551.19 3.09) at the same concentration
(Fig. 1b). The results of LPO and protein fragmentation experiments
showed that the EA fraction was an active scavenger of hydroxyl
radicals, such that LPO and protein oxidation were signicantly
prevented by presence of EA fraction.
To identify the mechanisms involved in EA fraction-mediated
antioxidant activity, and particularly to determine whether the
fraction inhibits hydroxyl radical generation by chelating metal
ions or by directly scavenging hydroxyl radicals, we used two dif-
ferent experimental approaches. In site-specic assay was em-
ployed to verify whether fraction was able to protect this
carbohydrate through metal chelation using ascorbic acidiron
H
2
O
2
EDTA system. On the other hand non-site-specic assay
was employed to verify fraction was able to direct scavenge
hydroxyl radical using without EDTA. As shown in Fig. 1c and d,
concentration-dependent inhibition of hydroxyl radical-induced
deoxyribose degradation was observed. Relatively greater antioxi-
dant activity was observed in the site-specic assay (92.44 4.56%)
than in the non-site-specic assay (84.51 4.41%) when the same
concentration 500 lg/ml of EA fraction was used, implying that EA
fraction chelates metal ions rather than scavenging hydroxyl radi-
cals directly. EA fraction is rich in quercetin has been well-known
natural antioxidant, which act as a strong metal ion chelator (Prak-
ash et al., 2007a; Sestili et al., 1998). Antioxidant activity values of
BHT in the site-specic assay was varied from 6.25 0.71% to
59.31 3.41% and 22.93 1.17% to 82.71 5.99% in non-site-spe-
cic experiment at the same concentration, which was lower than
EA fraction of red onion peel.
Superoxide anion is a well-recognized free radical species and is
generated continuously by auto-oxidation processes or by several
cellular processes, including the microsomal and mitochondrial
electron transport systems. Moreover, superoxide anion produce
other kinds of cell-damaging free radicals and oxidizing agents that
initiate DNA, proteins and lipids oxidation and implicated several
pathophysiological processes such as cancer, aging and neurode-
generative diseases (Fridovich, 1995; Yen and Duh, 1994; Patricia
et al., 2005). Therefore, we used the PMSNADHNBT system, a
non-enzymatic universal method. Fig. 1 e shows that EA fraction
inhibited NBT reduction very efciently. For example, EA fraction
inhibited the production of superoxide anion by 22.62 1.68%,
49.74 3.94%, 83.26 5.84%, 92.41 4.36% and 97.13 3.23%
when 100, 200, 300, 400 and 500 lg/ml of the extract were added
to the reaction mixture, respectively. The free radical scavenging
potential of EA fraction was further substantiated by scavenging
of nitric oxide radical, assayed by sodium nitroprusside method.
Incubation of sodium nitroprusside solution in phosphate buffer
saline at 25 C for 2 h at physiological pH spontaneously generates
nitric oxide which interacts with oxygen to produce nitrite ions
that can be estimated using a Griesss reagent. EA fraction showed
strong inhibitory effect on nitric oxide radical and values varied
from 21.64 1.76% to 93.92 4.34% in a concentration-dependent
manner (100500 lg/ml) are illustrated in Fig. 1f. EA fraction had
higher scavenging potential on both superoxide anion and nitric
oxide radical than standard BHT. Phytochemical analysis of EA
fraction indicated the presence of phenols and avonoids. These
phytomolecules exhibits range of biological properties toward can-
cer, cardiovascular diseases and Alzheimers disease, only by their
strong free radical scavenging and antioxidant activities (Shutenko
et al., 1999). Hence, the large amount of these phenolics contained
in EA fraction may cause its strong scavenging and preventive
capacity against superoxide anion and nitric oxide and hydroxyl
radicals.
3.2. Antimutagenic activity
The antimutagenic activity of EA fraction was studied on S.
typhimurium strain TA-102 by using Ames test against tobacco as
mutagen. Tobacco contains several potent mutagenic nicotin and
nor-nicotin substances (Barrett, 1991). Kuttan and Sukumaran
(1995) reported that tobacco extract produce signicant mutage-
nicity to this tester strain compared with other strains. In this
study, EA fraction was found to produce inhibition of mutagenicity
produced by tobacco in a concentration-dependent manner. At
concentration of 1000 lg of EA per plate, 70.41 4.79% of inhibi-
tion in the revertant colony formation was observed. Additions of
EA fraction at concentration of 200, 400, 600 and 800 lg per plate
have produced 10.72 1.12%, 20.59 1.39%, 32.77 3.96% and
53.27 4.47% of inhibition, respectively (Fig. 2). Mechanism of ac-
tion of EA fraction seems to be due to potent antioxidant activity of
the fraction and subsequent scavenging of ROS by the polyphenols
present in the extract. This is in agreement with the nding of
Santhosh et al. (2005) reported that green tea polyphenols inhibit
the tobacco extract-induced mutagenicity.
The antimutagenic activity of EA fraction was further conrmed
on pUC18 plasmid DNA as in vitro model system. Exposure of plas-
mid DNA to Fentons reagent ultimately results in strand breaks
formation, mainly due to the generation of reactive species-hydro-
xyl radical and the subsequent free radical-induced reaction on
plasmid DNA. Hydroxyl radical, react with nitrogenous bases of
DNA producing base radicals and sugar radicals. The base radicals
in turn react with the sugar moiety causing breakage of sugar-
phosphate back bone of nucleic acid, resulting in strand break for-
mation (Sonntag, 1987). For these studies, pUC18 plasmid DNA
was exposed to Fentons reagent in the absence and in the presence
0
10
20
30
40
50
60
70
80
200 400 600 800 1000
Concentration (g/plate)
%

i
n
h
i
b
i
t
i
o
n
a
d
e
c
b
Fig. 2. Antimutagenic effects of EA fraction to Salmonella typhimurium strains
(TA102) against tobacco extract as mutagen. Values are the mean SD (n = 3). Data
were analysed by ANOVA (
***
P < 0.001) and within each column different letters
indicate statistically different values according to post-hoc comparison (LSD-test) at
P < 0.01.
B.N. Singh et al. / Food and Chemical Toxicology 47 (2009) 11611167 1165
of EA fraction and the conversion of the native/supercoiled form to
open circular and linear form was monitored using horizontal gel
electrophoresis is presented in Fig. 3. Increase in open circular
and linear form due to oxidative damage provoked by hydroxyl
radical can be seen clearly in Lane 2. The plasmid DNA samples
were incubated with Fentons reagent in presence of EA fraction
to observe its effect on inhibiting strand breaks formation induced
by hydroxyl radical. Lane 48 of Fig. 3 showed signicant reduction
in the nicked circular and linear forms, increased to native/super-
coiled and mitigated the oxidative stress in the presence of differ-
ent concentrations at 210 lg/ml of EA fraction, respectively. This
EA fraction-mediated antimutagenic activity on plasmid pUC18
DNA was similar to that of 5 U of catalase used as accepted antiox-
idant enzyme (Lane 3). These results indicate that the EA fraction
effectively counteracts the oxidative stress produced by Fenton-
like reaction on susceptible biomolecule such as DNA.
Several polyphenolic rich extracts and puried compounds have
been reported for their potent antimutagenic activity and DNA pro-
tective ability (Santhosh et al., 2005; Hour et al., 1999; Prakash
et al., 2007a,b; Singh et al., 2009). Antimutagenic and oxidative
DNA damage protective activities of EA fraction may be due to
the action of polyphenols like chlorogenic acid (Shibata et al.,
1999), gallic acid (Abdelwahed et al., 2007; Prakash et al., 2007b)
and quercetin (Aherne and Brien 1999; Geetha et al., 2005; Jun
et al., 2007; Prakash et al., 2007a) which are present in extract of
red A. cepa peel.
As a conclusion, it can be stated that red onion peel as a good
and easily accessible source for nutraceutical compounds. For the
rst time the various extracts/fractions have been used to demon-
strate the antioxidant and antimutagenic activities of red onion
peel. Results showed that the methanolic fractions possessed sig-
nicant antioxidant and radical scavenging activities. More specif-
ically, the ethyl acetate fraction exhibited the best antioxidant and
free radical scavenging activities among the others and fraction
also shows signicant antimutagenic property. The high antioxi-
dant and antimutagenic activities of the ethyl acetate peel fraction
arising mainly from the presence of well-known antioxidant com-
pounds (i.e. polyphenols) in the large amount. We consider that
this peel fraction deserves more intensive study, including its
in vivo antioxidant activity, bioavailability and possible protection
against cardiovascular diseases to understand their real potential
as nutraceuticals.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgements
The authors are grateful to Director, National Botanical Re-
search Institute (NBRI), Lucknow, India, for his keen interest and
Department of Science and Technology (DST) New Delhi, providing
nancial support.
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