5 (2013) 1191 –1203

JOURNAL OF FUNCTIONAL FOODS

Antioxidative phenolic constituents of skins of onion varieties and their

activities
Tasahil Albishia, Jenny A. Johna, Abdulrahman S. Al-Khalifab, Fereidoon Shahidia,c,
*
aDepartment of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B 3X9 bDepartment of Food
Science and Nutrition, King Saud University (KSU), Riyadh, Saudi Arabia cDistinguished Visiting Professor, KSU, Saudi Arabia
ARTICLEINFO
Article history: Received 14 February 2013 Received in revised form 11 March 2013 Accepted 1 April 2013 Available online 1
July 2013
Keywords: Onion Green shoots Phenolic content Free radical scavenging activity HPLC–MS
ABSTRACT
The antioxidant activity of phenolic constituent of skin and selected flesh of different col- oured onions (Pearl, Red, Yellow and
White) were determined. The green shoot obtained after sprouting of the red onion was also analyzed to study the changes in the
phenolic constituents during germination. For the first time, all tests were carried out separately for the free, esterified and
insoluble-bound phenolic constituents of onion samples. The content of phenolics extracted from onion skins was approximately
six times higher than that of their flesh counterparts. Among onion varieties, pearl onion skin showed the high- est phenolic
content (26.4 mg quercetin eq/g freeze dried sample). A similar trend was observed for free radical scavenging activity of the
tested samples. Extracts from edible part of onion showed lower activity in all antioxidant tests carried out. The HPLC–MS
analysis showed that quercetin 3,40-diglucoside, quercetin, and kaempferol were the predominant phenolics in all onion extracts
tested.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Onions (Allium cepa), one of the most widely consumed vegeta- bles, are classified based on their colour into yellow, red and
white and based on their taste as sweet and non-sweet (Shahidi & Naczk, 2004). Fresh and dehydrated onions are widely used in
human diet as a source of nutrients, spicy garnish and non- nutritive health promoting compounds (Lee, Lee, Choi, Lee, & Levin,
2008). Onions contain a number of phytochemicals and their consumption has long been associated with health pro- motion and
disease risk reduction; decreasing the incidence of cancers in several tissues, preventing vascular and heart dis- eases,
neurodegenerative disorders and cataract formation (Kaur, Joshi, & Kapoor, 2009). Among phytochemicals with health benefit,
flavonoids, fructans and organosulphur com-
pounds in onions are considered important contributing fac- tors to the overall antioxidant activity of the diet (Ames,
Shigenagaand, & Hagen, 1993; Paganga, Miller, & Rice-Evans, 1999; Rice-Evans, Miller, & Paganga, 1997).
Epidemiological studies about major sources of antioxidant intake highlighted the importance of onions for high levels of
flavonols (Hertog, Feskens, Hollman, Katan, & Kromhout, 1993; Suh, Lee, Cho, Kim, & Chung, 1999). Despite the high inter-
est that onions have generated in the field of antioxidants, few studies have addressed the need to evaluate the antioxidant
capacity of different species of onion. Red, yellow, and white onions are, in fact, known to contain a large amount of flavo- nols;
the majority being glucose derivatives of quercetin and keampferol (Lee & Mitchel, 2011; Rhodes & Price, 1996; Sellappan &
Akoh, 2002; Shim, Yi, & Kim, 2011). Onion skins
* Corresponding author at: Department of Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada A1B
3X9.
E-mail address: [email protected] (F. Shahidi). 1756-4646/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jff.2013.04.002
Available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jff
also contain significantly higher content of flavonoids than the edible portion by around 2–10 g/kg (Suh et al., 1999). Regardless
of high levels of flavonoids in outer scales of onions, they are peeled off and discarded before food processing such as cook- ing.
The outer scales contain quercetin derivatives (Furusawa, Tanaka, Nakaya, Iinuma, & Tsuchiya, 2002; Furusawa et al., 2003;
Takahama & Hirota, 2000) which account for more than 80% of the total content of flavonoids in onions (Furusawa et al., 2002;
Furusawa et al., 2003; Galdo ́ n, Rodrıguez, & Romero, 2008). Takahama and Hirota (2000) have suggested that quercetin is
formed by the deglucosidation of its glucosides, followed by autoxidation to produce protocatechuic acid. Recently, some
anti-platelet and membrane-rigidifying flavo- noids have been isolated from outer scales of onion and identi- fied as quercetin,
quercetin dimers, and quercetin 4-glucoside (Furusawa et al., 2002; Furusawa et al., 2003). Furthermore, dry onion skin has a
different composition of quercetin deriva- tives compared to the fleshy scales where as much as 53% of total quercetin is present
in the free form (Wiczkowski, Nemeth, Bucinski, & Piskula, 2003). This high amount of quercetin in the outer layers of onion
bulbs is probably a conse- quence of exposure to sunlight (Higashio, Hirokane, Sato, Tokuda, & Urgami, 2005; Lee et al., 2008).
Extracts from onion skins have exhibited potent radical scavenging activities (Nuutila, Kammiovirta, & Oksman-Caldentey,
2002); however, the specific antioxidative components are still unclear.
Although there have been extensive studies on the free phenolics and their antioxidant activities in onions, there ap- pears to
be very little information available in the existing lit- erature on their esterified and bound phenolics. This may lead to
underestimation of the total phenolics present and their contribution to the overall antioxidant activity. In this study, the phenolic
constituents of onion skin and flesh were fractionated into their respective free, esterified, and bound forms by alkali hydrolysis
before analysis. The green shoot from one of the sprouted onions was also evaluated along with the flesh to understand the
changes that may take place in the phenolic constituents during germination. The study aimed to provide information about the
potential use of onion skin extracts as an effective source of antioxidants in food systems; specifically to compare four different
varieties of onions with respect to their total phenolic content and anti- oxidant activity and the identity of the phenolics present.
2. Materials and methods
2.1. Materials
Onion samples, namely Pearl, Red, Yellow, and White varie- ties were purchased from a local market in St. John’s, NL, Can-
ada. Some of the red onions were allowed to sprout to test the phenolic composition of the sprouted red onion flesh.
Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from Acros Organics (Fair Lawn, NJ,
USA). Organic solvents and reagents such as methanol, acetone, and sodium carbonate were purchased from Fisher Scientific
Co. (Nepean, ON, Canada). The compounds 2,20-azobis (2- methylpropionamidine) dihydrochloride (AAPH), 2,20-azino-
bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), Folin and
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Ciocalteau’s phenol reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and all phenolic standards with purity (P96%) were ob-
tained from Sigma–Aldrich Canada Ltd. (Oakville, ON, Canada).
2.2. Sample preparation
Onions were peeled manually, and skin samples were freeze dried for around 3 days at À48 °C and 30 · 10À3 mbar (Freez- one
6, model 77530, Labconco Co., Kansas City, MO, USA). The flesh of red onion and sprouted red onion was also sepa- rated, cut
and freeze dried. The dried samples were then fi- nely ground to pass through a 0.5 mm sieve, vacuum packed and stored in a
freezer at À20 °C until analyzed. All experi- ments were carried out in triplicates and the results were re- ported as
means±standard deviation. The methodologies followed are described below.
2.3. Extraction of phenolic fractions
Free, esterified, and insoluble-bound phenolic compounds were extracted and fractionated as described by Krygier, Sosulski, and
Hogge (1982). Freeze dried onion skins and flesh samples (5g) were ultrasonicated for 20min at 30 °C with 150 mL of a mixture
of methanol–acetone–water (7:7:6, v/v/ v) The resulting slurries were centrifuged at 4000·g (ICE Cen- tra MS, International
Equipment Co., Needham Heights, MA, USA) for 5 min and the supernatants collected. The residue was re-extracted under the
same conditions. After centrifuga- tion, the combined extracts were analyzed for free phenolic acids and soluble phenolic esters,
and the residue was re- served for determination of insoluble-bound phenolic acids. The combined supernatants were evaporated
under vacuum at 40 °C to remove the organic solvents, and the aqueous phase was adjusted to pH 2 before extraction with
hexane to remove interfering lipids (Krygier, Sosulski, and Hogge, 1982). The free phenolic acids were then 4 times extracted
with diethyl ether–ethyl acetate (1:1, v/v), dried under vac- uum using a rotary evaporator and the extract was dissolved in 5 ml
of 80% methanol (HPLC grade). The esters remaining in the aqueous phase were hydrolyzed with 4 M NaOH and the liberated
phenolic acids were extracted with diethyl ether– ethyl acetate, dried and dissolved in 5 mL methanol as in the case of free
phenolics. The residues were initially dispersed in 50ml of 4M NaOH and stirred for 4 h under nitrogen. The solution was then
acidified slightly (pH 2), centrifuged and the bound phenolics were extracted with diethyl ether– ethyl acetate (1:1, v/v) as
described for esterified phenolics.
2.4. Chlorophyll removal
Studies show that chlorophyll is responsible for a pro-oxidant activity under light in food extracts. The pro-oxidant and
antioxidant properties of chlorophylls and their derivatives depend on the presence of light; when in dark medium chlo- rophylls
and their derivatives act as antioxidants, but under light, they act as photosensitizer pro-oxidants. Thus chloro- phylls were
removed from green shoot that sprouted from red onion to prevent interference by cholorophylls in the anti- oxidant tests.
Dechlorophyllization of green shoot extracts was carried out using liquid–liquid extraction according to
5 (2013) 1191 –1203
Alvarez-Parrilla, de la Rosa, Amarowicz, and Shahidi (2011). The crude phenolic extracts (1.5 g) were dissolved in 50 mL of
80% methanol and poured into an extraction funnel. Twenty-five millilitres of CH
2
Cl
2
were added; the separatory funnel was shaken and allowed to stand for phase separation. The
organic phase was removed, and extraction was repeated one more time. Methanol was partially removed under vac- uum at 45
°C, and the concentrated slurries were freeze-dried for 72 h at À45 °C. Dried extracts were stored at À20 °C. The yield of each
extract was then determined.
2.5. Estimation of total phenolic content
The total phenolic content was determined according to an improved version of the procedure explained by Singleton and Rossi
(1965). The content of total phenolics in each ex- tract was determined using a standard curve prepared for gal- lic acid and the
results were expressed as mg of gallic acid equivalents (mg GAE) per gram of dried onion peel/flesh.
2.6. Determination of total flavonoid content
Total flavonoid content was measured by the aluminum chlo- ride colorimetric assay (Zhishen, Mengcheng, & Jianming, 1999).
One millilitre of extracts or standard solution of querce- tin (0.75, 1.5, 3 mg/mL) was added to 10 mL volumetric flask containing
4 mL distilled water. To the flask, 0.3 mL of 5% NaNO
2
was added. After 5 min, 0.3 mL 10% AlCl
3
was added. After 6 min, 2 mL 1 M NaOH solution were added and the total
volume was made up to 10 mL with distilled water. The solution was mixed well and the absorbance was measured against a
prepared reagent blank at 510nm. Total flavonoid content was expressed as mg quercetin eq (QE)/g dry plant material.
2.7. Determination of total anthocyanin content
The content of anthocyanins was determined by the pH-dif- ferential method (Giusti & Wrolstad, 2001). Each extract (0.5 mL)
was diluted with 2.5 mL of 0.025 M potassium chlo- ride buffer, pH 1.0 and 0.4 M sodium acetate buffer, pH 4.5, separately. The
diluted solutions were then left at room tem- perature for 15 min, and the absorbance of each dilution was read at 520 and 700 nm
against a blank cell filled with distilled water. The anthocyanin content was calculated using the fol- lowing equation:
Anthocyanins content ðmg=100 g of dry matterÞ
1⁄4 A Ã MW Ã DF=ð Ã WÞ
where A = absorbance (A
520nm
ÀA
700nm
)
pH1.0
À(A
520nm
-ÀA
700nm
)
pH4.5
, MW = molecular weight of cyaniding-3-gluco- side (C
15
H
11
O
6
, 449.2), DF = dilution factor, e = molar absorptivity (26900), and W = sample weight (g).
2.8. In vitro antioxidant assays
2.8.1. Total antioxidant capacity by trolox equivalents antioxidant capacity (TEAC) The TEAC assay is based on scavenging of
2,20azinobis-(3-eth- ylbenzothiazoline-6-sulphonate) radical cation (ABTSÅ+) and was determined according to the method
described by Van
JOURNAL OF FUNCTIONAL FOODS
den Berg, Haenen, Van den Berg, and Bast (1999) and modified by John and Shahidi (2010). A solution of ABTSÅ+ was
prepared in 0.1 M phosphate buffer saline (pH 7.4, 0.15 M sodium chlo- ride) (PBS) by mixing 2.5 mM AAPH with 2.0 mM
ABTS stock solution at a 1:1 (v/v) ratio. The solution was heated for 12 min at 60 °C, protected from light and stored at ambient
temperature. The radical solution was used within 3h as the absorbance of the radical itself depletes with time. Potato extracts
were dissolved in PBS and diluted accordingly in or- der to fit in the range of values in the standard curve prepared using
different concentrations of trolox. To determine the TEAC values for extracts, 40 lL of samples were mixed with 1.96 mL of
ABTS·+ solution. Absorbance of the above mixture was read at 734 nm after 6 min as the extracts needed a min- imum of 6 min
for completion of the reaction; the decrease in the absorption was then used for calculating TEAC values. Blank measurements of
ABTSÅ+ stock solution were also made. TEAC values were expressed as micromoles of trolox eq (TE) per gram of dried onion
peel/flesh. TEAC values were determined as follows: DA
Trolox
1⁄4 A
trolox solution after 6 min
ÀA
blank after 6 min
DA
extract
1⁄4 A
extract after 6 min
ÀA
blank after 6 min
DA
Trolox
1⁄4 m  1⁄2Trolox​
TEAC
extract
1⁄4 ðDA
extract
=mÞ Â d
where DA = reduction of absorbance, A = absorbance at a given time, m = slope of the standard curve, [Trolox] = concen-
tration of trolox, d = dilution factor.
2.8.2. DPPH radical scavenging capacity (DRSC) using electron paramagentic resonance (EPR) DRSC assay was carried out
using the method explained by Madhujith and Shahidi (2006). Two millilitres of 0.18 mM solu- tion of DPPH in methanol were
added to 500 lL of appropri- ately diluted free, esterified and bound phenolics extracts in methanol. Contents were mixed well,
and after 10min, the mixture was passed through the capillary tubing which guides the sample through the sample cavity of a
Bruker e- scan EPR spectrophotometer (Bruker E-Scan, Bruker Biospin Co., Billercia, MA, USA). The spectrum was recorded
on Bruker E-scan food analyzer (Bruker Biospin Co.). The parameters were set as follows: 5.02 · 102 receiver gain, 1.86 G
modulation amplitude, 2.621s sweep time, 8 scans, 100.000G sweep width, 3495.258 G centre field, 5.12 ms time constant, 9.795
GHZ microwave frequency, 86.00 kHZ modulation fre- quency, and 1.86G modulation amplitude. For quantitative
measurements of radical concentration remaining after reac- tion with the extracts, the method of comparative determina- tion
based on the corresponding signal intensity of first-order derivative of absorption curve was used. DRSC of the extracts was
calculated using the following equation:
DPPH radical scavenging capacity;% 1⁄4 100
À ðEPR signal intensity for the medium containing the extract =EPR signal intensity for the control mediumÞ Â 100:
From the standard curve plotted for the DRSC of trolox, the scavenging activity of potato extracts was determined and ex-
pressed as lmol TE/g dried onion peel/flesh.
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1193
2.8.3. Determination of reducing power The reducing power of the extracts was determined as de- scribed by Amarowicz,
Karamac, Weidner, Abe, & Shahidi, (2002). A standard curve for trolox was constructed and re- sults were expressed as lmol of
TE/g dried onion peel/flesh.
2.8.4. Measurement of iron (II) chelating capacity The chelation of ferrous ion by the extract was estimated by the method of
Liyana-Pathirana and Shahidi (2006) with some modifications. In brief, 0.5mL of extract was mixed with 1.85mL of methanol
and 0.05mL of 1mmol/L ferrozine, followed by vigorous shaking and allowing the mixture to react at room temperature for
10min. The absorbance was measured spectrophotometrically at 562 nm. The chelation capacities of onions were expressed as
lmol ethylenediaminetetraacetic acid (EDTA) eq/g extract. The blank was devoid ferrozine. Iron chelation capacities of the
extracts were calculated using the following equation:
Fe ðIIÞ chelation capacity;% 1⁄4 ð1 À Absorbance of extractÞ
Ã100=Absorbance of blank=EPR signal intensity for the control medium
2.9. Determination of 2-thiobarbituric acid reactive substances (TBARS) in cooked comminuted fish meat model system
Ground salmon that was used for the determination of TBARS was first analyzed for its moisture and fat contents. The mois- ture
content was calculated as the percent ratio of the weight difference of the sample before and after drying at 100 ± 1 °C to that of
the original material (AOAC, 1990). Total fat content of the samples was determined using the procedure described by Bligh and
Dyer (1959) and modified by Shahidi (2001).
Fish meat model systems were prepared as described by Shahidi and Pegg (1990). Ground salmon (80g) was mixed with
deionized water (20 mL) in Mason jars. Soluble extracts of onion (200ppm based on phenolics content) as well as 200 ppm BHA
and quercetin, respectively, were added sepa- rately to the mixture in the Mason jars and thoroughly homogenized. A control
sample containing no extract was also prepared. Samples were cooked in a thermostated water bath at 80 ± 2 °C for 40 min while
stirring every 5 min with a glass rod. After cooling to room temperature, fish samples were homogenized for 30s, transferred into
plastic bags, and then stored in a refrigerator at 4 °C for 7 days. Samples for the analyses of TBARS were drawn on days 0 and 7.
TBARS were determined using a modified version of the method described by Wijeratne, Abou-Zaid, and Shahidi (2006).
Two grams of each sample were weighed in a centrifuge tube to which 5 mL of a 10% (w/v) solution of TCA were added and
vortexed (Fisher Vortex Genie 2; Fisher Scientific, Nepean, ON, Canada) at high speed for 2 min. An aqueous solution (0.02 M)
of TBA (5 mL) was then added to each centrifuge tube, followed by further vortexing for 30 s. The samples were sub- sequently
centrifuged at 3000·g for 10 min and the superna- tants were filtered through a Whatman No. 3 filter paper. Filtrates were heated
in a boiling water bath for 45 min, cooled to room temperature in cold water, and the absorbance of the resultant pink-coloured
chromogen read at 532 nm. A stan-
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dard curve was prepared using 1,1,3,3-tetramethoxypropane as a precursor of malondialdehyde (MDA; 0,1,2,3 and 6 ppm). The
TBARS values were then calculated using the standard curve and expressed as milligrams MDA eq/kg of sample.
2.10. High pressure liquid chromatography (HPLC) analysis of phenolic compounds
The identification of phenolics was performed using HPLC with photodiode array detection (DAD) according to the pro- cedure
originally described by Escarpa and Gonzalez (2000) using an Agilent 1100 Series HPLC system (Agilent Technolo- gies, Palo
Alto, CA, USA) equipped with a quaternary pump (model G1311A), a degasser (model G1379A), an autosampler (automatic
liquid sampler, ASL, model G1329A) and a diode array detector (model G1315B DAD) linked to an HP-ChemSta- tion data
handling system. Twenty-five microlitres of the sample extracts were automatically injected into a prepacked Supelcosilä LC-18
column (250 · 4.6 mm inner diameter, 5-lm particles, Supelco, Bellefonte, PA, USA) at room temperature. Phenolics were
detected at 280 and 325 nm. The elution sol- vents were A (aqueous 0.01 M phosphoric acid) and B (100% methanol). The
samples were eluted according to the follow- ing binary gradient: starting with 5% B in order to reach 50% B at 10 min, 70% B at
15 min, 80% at 20 min and 100% at 25 min. The flow rate was 1 mL/min. Compounds were identified and quantified by
conventional retention times using appropriate standards.
For HPLC–MS analysis, an Agilent 1100 SL LC/MSD ion trap mass spectrometer (Agilent Technologies, Palo Alto, CA,
USA) was connected to the Agilent 1100 HPLC system via an ESI interface in the negative ion detection mode. The MS revealed
the negative molecular ions; MS–MS broke down the most abundant one with dependent collision-induced dissociation. The
selected values for spray chamber parameters were as follows: capillary potential, 3500 V; gas temperature, 350 °C; drying gas
flow, 13 L minÀ1; nebulizer pressure, 414 kPa. For full scan MS analysis, the spectra were recorded in the range of m/z 50–700.
Identification of compounds by HPLC–MS anal- ysis was carried out by comparing retention times and mass spectra of the
unknown peaks to those of the standards.
2.11. Statistical analysis
Statistical analysis was performed using SigmaStat version 10.0 (Jandel Corp., San Raphael, CA, USA). Results were sub- jected
to ANOVA, and differences between means were lo- cated using Tukey’s multiple comparison test. Correlations between various
parameters were also investigated. Signifi- cance was determined at p 6 0.05 level. All data were reported as the mean ± SD for
three replications.
3. Results and discussion
3.1. Total phenolic content
Preliminary experiments revealed that the mixture of metha- nol–acetone–water (7:7:6, v/v/v) was the best solvent for the
extraction of phenolics from onion, either flesh or skin, when
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ultrasonicated for 20 min at 30 °C to afford a maximum yield of phenolics (Results not shown).
The total free, esterified and insoluble-bound phenolics were determined, for mthe first time, in onion samples which included
pearl onion as well as sprouted onion. The content of free phenolics in tested onion varieties was, in decreasing order, pearl onion
skin>red onion skin>yellow onion skin>red onion flesh>sprouted red onion flesh>white onion skin (Table 1). The esterified and
bound forms of pheno- lic compounds in the onion samples followed a similar trend. White onion skin extract had the lowest
amount of free, esterified, and bound phenolics compared to the others onion varieties (Table 1). There was no significant (p >
0.05) differ- ence between free phenolic content of red and yellow onion skin or between the non-sprouted and sprouted red
onion flesh. However, bound phenolic content in yellow onion skin was significantly (p < 0.05) lower than that in red and pearl
onion skins (Table 1).
Phenolics were predominantly present in the free form both in the onion skin and flesh, except in red onion skin where the
bound phenolics were present in higher quanti- ties (41.30%) than the free form (38.41%). The percentage of free phenolics in
the total phenolics of pearl, red, yellow, white skin, red flesh, sprouted red flesh and green shoot were 56.85, 38.42, 44.17, 70.5,
90.17, and 95.14%, respectively. The bound phenolics were present in higher concentrations (41.30%) than the esterified forms
(20.28%) in the skin; while the flesh had higher concentrations of the esterified (9.67%) as compared to the bound form (0.73%)
of phenolic com- pounds. However, the free phenolics content of white onion skin was 32 times lower than those present in red
onion flesh. Similar results were reported by Prakash, Singh, and Upadhyay (2007), though they studied only the free pheno- lics
of different onion varieties. They reported the TPC of the skin and flesh of red onions as 74.1 and 21.5 mg GAE/g dried onion
which are higher than those obtained in the present study (62.6 and 18.04mg GAE/g sample). The TPC for white onion was also
reported to be much lower than the red variety. Shon, Choi, Kahng, Nam, and Sung (2004) also reported the phenolic compound
and antioxidant activity of red onion to be higher than those of yellow and white onions.
The phenolic content in the red onion skin and flesh were analyzed to determine the distribution of phenolics in onions in
general. The red onions were also sprouted to study the changes in the phenolics during germination and its content in the green
shoot that emerges during germination. As ex- pected, the phenolic composition of the red onion flesh was 1.4 times lower than
those found in the surrounding skin but higher than in the sprouted red onion flesh samples and green shoots. It is also interesting
that the free and esterified forms of phenolics were present at a higher concentration in the red flesh, the sprouted flesh; green
shoot had higher con- tent of bound phenolics. It is clear that the phenolics in the flesh decreased during germination. Though no
such similar studies have been done for onions, Tian, Nakamura, and Kayahara (2004) and Tian, Nakamura, Cui, and Kayahara
(2005) studied the changes in phenolic constituents of brown rice during germination. They observed a reduction of
approximately 70% in the concentration of feruloylsucrose
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1195
and sinapoylsucrose, with an increase in the content of ferulic and sinapic acids in the light brown pericarp of rice grains during
germination. They speculated that this reduc- tion was probably caused by the hydrolysis, indicating that germination caused the
metabolism of phenolic compounds. In addition, the total content of insoluble phenolic com- pounds increased from 18.47
mg/100 g of flour in brown rice to 24.78 mg/100 g of flour in germinated brown rice, similar to that observed for sprouted
onions. In the cell wall, phenolic compounds, particularly hydroxycinnamates, are ester linked to insoluble fibres,
polysaccharides, and lignin components. The increase in phenolic compounds during germination could be explained as an
increase in the free forms with alka- line hydrolysis, due to dismantling of the cell walls during germination. It is speculated that,
during germination, as product moisture increases, there is a potential for injury by oxidation and/or microorganism infiltration.
Induced saccha- rolytic enzymes to hydrolyze starch would produce free phe- nolic compounds having more effective antioxidant
activity than hydroxycinnamate sucrose esters. As a result, the con- tent of hydroxycinnamate sucrose esters decreased, whereas
that of free phenolic compounds increased (Tian et al., 2004). This may be due to the changes taking place in the sprouted red
onion flesh; however this explanation requires experi- mental verification.
3.2. Total flavonoid content
In vitro and in vivo studies have demonstrated that flavonoids exhibit a variety of biological activities including antioxida- tive
effects (Boyle et al., 2000), reduction of cardiovascular dis- ease (Janssen et al., 1998) and reduction of the risk of rheumatoid
arthritis (Pattison et al., 2004). Flavonoids are an important group of compounds present in large amounts in onions (Prakash et
al., 2007).
In the present study, red onion skin exhibited the highest free flavonoid content (20.22±0.39mg/g sample), followed by pearl
onion skin (19.64 ± 0.2 mg/g sample). However, there was no significant difference (p > 0.05) between the free, ester- ified and
bound flavonoids of red and pearl onion skin. White onion skin contained the lowest free flavonoids content (0.08±0.08mg/g).
Bonaccorsi, Caristi, Gargiulli, and Leuzzi (2008) also made a similar observation and found that the fla- vonol content in white
onion bulb was about 7 mg/kg as com- pared to 600–700 mg/kg in red and gold onion varieties. It is also clear that the free
flavonoids were present in highest quantities in all varieties compared to the esterified and bound flavonoids (Table 1). As
expected, the flavonoid content in red onion skin was also higher than those in red onion flesh. Price, Bacon, and Rhodes (1997)
have also indicated that the outer layers of onions are rich in flavonoids compared to the whole onion bulb or edible parts.
Moreover, there is evi- dence of decreasing trend in the content of some flavonoids from the dry skin to the inner rings (Patil &
Pike, 1995). Mean- while, USDA Nutrient Data Laboratory. (2007) reported that the range of quercetin in different onions varied
from 33.43 ± 2.38 mg/g in red onion bulb to 7.29 ± 1.27 mg/100 g in young green onion. These results are similar to those of the
present study for free form of phenolics in sprouted and reg- ular red flesh onions (Table 1).
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Among the different varieties of onion, the highest level of quercetin has been reported in fleshy scales of yellow/brown onion
(170–1200mg/kg fresh weight) and in red onion (190–1900 mg/kg fresh weight), while lower levels of quercetin were found in
white onion (50–650mg/kg fresh weight) (Crozier, Lean, McDonald, & Black, 1997; Lugasi & Hovari, 2000; Price & Rhodes,
1997; Tsushida & Suzuki 1996). The compounds quercetin-40-glucoside and quercetin-3,40- diglucoside are two main
derivatives of quercetin found in red onions (Leighton et al., 1992). High concentration of quer- cetin has been found in dry red
onion skin. This part of onion contains fivefold higher content of quercetin in comparison to the flesh layers (Patil & Pike, 1995).
Dry onion skin also has a different content of quercetin derivatives compared to the flesh layers where as much as 53% of total
quercetin was present in the free form (Wiczkowski et al., 2003). This high amount of quercetin in the outer layers of onion bulb
is probably a consequence of exposure to sunlight that may promote rapid synthesis of quercetin after harvest (Higashio et al.,
2005; Lee et al., 2008).
3.3. Total anthocyanin content
Total anthocyanins content was determined in the soluble phenolics extracts as mg cyanidin-3-O-glucoside eq. per gram of dry
onion peel/flesh. Anthocyanins in different varieties of onion in the order of their decreasing content were red onion skin > pearl
onion skin > red onion flesh > sprouted red onion flesh > yellow onion skin > white onion skin > green shoots (Table 1). This is
in agreement with cited data from Gorinstein et al. (2008) who reported that white onion had a low content of anthocyanins
compared to coloured onions, both for skin and flesh. The highest content of anthocyanins was present in red onion skin (10.04 ±
0.90 mg/100 g skin), while anthocy- anin content in white onion skin was 0.06 ± 0.01 mg/100 g. This result is in agreement with
that of Lauro and Francis (2000) who reported that the total anthocyanins in red onions was 7–21 mg/100 g sample. There was
also no significant dif- ference (p > 0.05) between green shoot of sprouted red onion and white onion skin.
More than 20 derivatives of anthocyanins have been iden- tified in red onions (Slimestad, Fossen, & Va ̊gen, 2007). Cyanidin
glucosides and acylated glucosides of cyanidins are the main anthocyanins of red onions (Donner, Gao, & Mazza, 1997; Fossen,
Andersen, Ovstedal, Pedersen, & Raknes, 1996). Moreover, cyanidin-3-(600-malonyl)-glucoside repre- sents more than 50% of
the total anthocyanins in different cultivars of red onion, and there is 20–250 mg/kg of anthocy- anins in fresh weight of red
onion (Fossen et al., 1996). Anthocyanins constitute about 10% of the total flavonoid con- tent of red onions (Rhodes & Price,
1996). Makris (2010) has reported that the utilization of waste from more pigmented onions as well as consideration of factors
such as tempera- ture and particle size could make onion wastes as a promising source of anthocyanins as water-soluble
pigments. Rhodes and Price (1996) found that anthocyanins are heavily concen- trated in the skin and in the outer fleshy layers,
whereas in the edible tissue they are restricted to a single layer of cells in the epidermal tissue. Gennaro et al. (2002) reported that
the dry skin of onions is rich in anthocyanins and flavonols,
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JOURNAL OF FUNCTIONAL FOODS
with high percentage of glycone forms that corresponds to 2% of the total weight in the portion that cannot be eaten and is
generally discarded. Therefore, $63% of total red onion anthocyanins are present in the dry skin and outer fleshy lay- ers that
accounts for 15% of the total weight. Ferreres, Gil, and Toma ́s-Barbera ́n (1996) detected cyanidin 3-glucoside and cyanidin
3-arabinoside and their malonated derivatives in red onions. Therefore the skin of coloured onions can serve as an excellent
source of natural cyanidin derivatives.
3.4. Antioxidant activity assays
Although a wide range of model systems are available for evaluation of the potency of antioxidant substances, the choice depends
mainly upon the chemical nature of the con- stituents. There is evidence of discrepancies in antioxidant activities of substances
when tested in different model sys- tems (Wettasinghe & Shahidi, 1999). Taking this into consider- ation, in the present study,
the antioxidant activities of the extracts were measured using several assays, including TEAC, DRSC, reducing power and iron
chelation.
3.4.1. TEAC assay The TEAC method is frequently used for determination of antioxidant activity. TEAC assay is based on the
scavenging of 2,20-azinobis-((3-ethylbenzothiaoline-6-sulphonate) radical cation (ABTS·+) by the antioxidant over a period of 6
min. The TEAC values of extracts were determined and compared with trolox. Trolox reacts instantly with ABTSÅ+ and the
reaction is completed within one minute. However, due to the biphasic nature of most antioxidative compounds, 6min which in-
cludes a greater part of the slow-biphasic reaction (Van den Berg et al., 1999) has shown good results. Therefore, 6 min was used
as the time point in the present study.
Generally, samples with higher phenolic content were most effective as free radical scavengers. The free phenolics in the skin
showed higher activity than the esterified and bound phenolics in the skin and flesh as was the trend for their pheno- lic content.
Table 2 lists the TEAC values of skin and flesh ex- tracts of onions. Though pearl onions had higher free phenolic content than
red and yellow onions, they showed low- er TEAC activity possibly because of the existing difference in the chemical nature of
their phenolic constituents and hence activity differences. Esterified extracts showed low TEAC val- ues, ranging from 0.05
(white skin) to 1.92 (pearl skin) mmoles TE/g dried sample. TEAC values of bound phenolic extracts var- ied from 0.04 mmoles
TE/g for white onion skin to 3.40 mmoles TE/g for red onion skin. Red onion skin had the highest TEAC value, 384.25 times
higher than that of white onion skin in the free, esterified, and bound phenolics. TEAC method is use- ful in screening
antioxidants, but antioxidant effectiveness must also be studied by other methods because their activity in foods is dependent on a
variety of factors, including polarity, solubility, metal-chelating capacity and the system used for their evaluation. Others have
measured the TEAC activity of different onions, (though not separately studying the activity of the free, esterified and bound
phenolics) as 15.6 (Sellappan & Akoh, 2002), 29.02 (Bahorun, Luximon-Ramma, Crozier, & Aruoma, 2004) and 64.11 lM TE/g
of DW (Proteggente et al., 2002).
5 (2013) 1191 –1203
1197
3.4.2. DPPH radical scavenging capacity (DRSC) Red onion skin showed the highest DRSC, followed by yellow onion skin,
pearl onion skin, red onion flesh, sprouted red onion flesh and white onion skin (Table 2). The trend is simi- lar to those obtained
for the phenolic and flavonoid contents of the samples and clearly indicating that samples with high- er phenolic content showed
higher antioxidant activity. How- ever, pearl onion skin that had a higher phenolic content than red onion skin showed a slightly
lower, although not signifi- cant (p > 0.05), DRSC; possibly because the chemical constitu- ents contributing to the scavenging
activity may be somewhat different. Velioglu, Mazza, Gao, and Oomah (1998) and Shahidi and Naczk (2004) reported that the
antiox- idant activity of a given food or food product depends on the chemical nature of its constituents and, not always their
quantities, as the efficacies of compounds present varies con- siderably. Furthermore, DRSC of the sprouted red onion flesh was
mainly contributed by its esterified phenolics, while in the red onion flesh free phenolics displayed the highest activ- ity. It is also
interesting that the free phenolics in the onion samples are the strongest DPPH radical scavengers as com- pared to the bound and
esterified forms except in the case of white onion peels and sprouted red onion flesh, where the esterified phenolics were more
abundant and had a high- er activity. In the DRSC assay, the onion extracts were able to reduce the stable DPPH radical to the
yellow coloured diph- enylpicrylhydrazine. Thus, the DRSC of onion extracts may be mostly related to their phenolic hydroxyl
group. Red onion skin extracts possessed the highest activity (more than 5.63 times higher than those of red onion flesh), the red
onion being again more active than the yellow onion. Prakash et al. (2007) and Gorinstein et al. (2008) also found a similar trend
in the DRSC of red onion skin and flesh.
3.4.3. Reducing power activity Reducing power of the onion extracts was determined by the method of Amarowicz et al. (2002).
Compounds that have reduction potential, react with potassium ferricyanide (Fe3+) to form potassium ferrocyanide (Fe2+) which
then reacts with ferric chloride to form a complex that has an absorption max- imum at 700nm. These compounds with reducing
power indicate that they are electron donors and can reduce the oxi- dized intermediates of lipid peroxidation processes, so that
they can act as primary and secondary antioxidants (Jayanthi & Lalitha, 2011).
Table 2 shows the reducing power of the free, esterified and bound phenolics of the extracts using the potassium fer- ricyanide
reduction method. The reducing power of the differ- ent varieties followed the same trend as other antioxidant activity tests with
free phenolics of pearl onion skin showing the highest (3.58 ± 0.01 mmoles TE/g sample) and white onion skin the lowest
activity (1.36±0.07mmoles TE/g sample). However, the reducing power of the bound phenolics was comparable to that of the
free phenolics, unlike those for DPPH and TEAC assays. In general, it can be concluded that free phenolics are the dominant
form of phenolics and con- tribute most to the antioxidant activity of onions followed by bound and esterified phenolics. There
was no significant difference between free phenolics of red pearl onion skin and yellow onion skin.
1198
JOURNAL OF FUNCTIONAL FOODS
3.4.4. Iron (II) chelating activity Metals such as iron, copper, manganese, nickel and cobalt at their higher valence state are
known to participate in direct initiation of lipid oxidation via electron transfer and lipid al- kyl radical formation while lower
valence metals can directly initiate lipid oxidation via the formation of reactive oxygen species (ROS) (Kanner, Harel, & Hazan,
1986). So, chelation of free iron can prevent the formation of free radicals as well as preventing the impairment of vital organ
function in vivo. The formation of a complex is formed between antioxidant and the metal renders metal ions inactive so that they
cannot any longer act as initiator of lipid oxidation (Shahidi & Zhong, 2007).
In the determination of iron chelating capacity of onion extracts, the iron chelating activity of free phenolics of onion skin,
expressed as lmol EDTA eq/100g sample, indicated very strong activity. Additionally, the free phenolics of pearl onion skin
showed the highest iron chelating activity (2991.45 ± 403.30 lmoles EDTA eq/100 g dried onion), although not significantly
different from that of red and yellow onion skin. As expected the extracts from the skin exhibited higher activity than those from
the red onion flesh and white onion skin. There were no significant differences among the iron chelating activities of white onion
skin, green shoot and red onion flesh samples. These results, presented in Table 2, fol- low the same trend as those observed for
other antioxidant activity tests employed in this study.
Phenolic compounds are the major fractions that chelate metal ions although non-phenolic constituents in the crude extracts
may also participate in sequestering of metal ions (Wettasinghe & Shahidi, 2002). Onions are a rich source of flavonoids which
can effectively deactivate prooxidant metal ions and thus prevent or retard metal ion-induced lipid oxida- tion. Quercetin, a
dominant flavonoid in onions is well known as a strong metal ion chelator (Prakash et al., 2007).
In general, it can be concluded that free phenolics are the dominant form of phenolics and the highest contributors to the
antioxidant activity of onions; pearl and red onion skins exhibiting the highest activity among all the varieties tested.
3.5. Inhibition of oxidation in fish model system
During lipid oxidation, malondialdehyde (MDA), a minor sec- ondary oxidation product of fatty acids with 3 or more double
bonds, is formed. MDA reacts with 2-thiobarbituric acid (TBA) to form a pink TBA-MDA complex that is measured spectro-
photometrically at its absorption maximum at 532 nm (Antol- ovich, Prenzler, Patsalides, McDonald, & Robards, 2002; Shahidi
& Zhong, 2007).
The muscle of salmon used for the analysis contained 12.73 ± 0.27% total lipids. This is in agreement with that re- ported by who
reported that mean fat content in salmon was 12.4–17.9%. The moisture content in salmon was 62.18 ± 0.65%. The TBARS
values of antioxidant-treated fish meat sam- ples stored at 4 °C over 7 days are shown in Table 3. The sol- uble onion extracts
were added at 0.1% and the reference antioxidants, BHA and chlorogenic acids were each added at 200 ppm. The extracts were
effective in inhibiting the oxida- tion of cooked salmon in comparison with the control, which showed the highest TBARS values
at the end of the 7 days of
5 (2013) 1191 –1203
ent Table 3 – Effect of extracts from onion samples on the formation of TBARS in a cooked fish model system.1
in the onion samples that were identified and quantified (mg/g dried onion sample) using HPLC are listed in Tables 4.
Quercetin, quercetin 3,40-diglucoside and kaempferol were Soluble extracts added to pork2
TBARS (mg of MDA eq/kg fish)
predominant in the free form in all onion samples; quercetin Day 0 Day 7
glucoside being the most abundant (Fig. 1).
Control Quercetin 2.40 ± 0.05a 5.01 ± 0.56a 2.66 ± 0.59a 3.17 ± 0.81b
Quercetin and kaempferol could easily be identified with standards. However, quercetin 3,40-diglucoside was identified
BHA 1.43 ± 0.06b 2.41 ± 0.37c
by its mass spectral data. The use of fragmentation in mass
Pearl onion skin 0.20 ± 0.08c 1.98 ± 0.09d
spectrometry allowed us to observe the corresponding
flavo- Red onion skin 0.14 ± 0.05d 1.58 ± 0.40e
nol aglycone as fragments of the molecular ion. Quercetin
White onion skin 0.19 ± 0.23e 2.68 ± 0.53f Yellow onion skin 0.87 ± 0.04f 2.82 ± 0.45g Red onion flesh 2.55 ± 0.27a 2.49 ±
0.05h Red onion flesh, sprouted 2.40 ± 0.06
a
4.52 ± 0.32
a
Green shoot 2.74 ± 0.16a 2.34 ± 0.20i
3,40-diglucoside at retention time, 19.5 min showed molecular ion [MÀH]À with m/z value of 625. The fragmentation (MS2) in
negative mode of the ion with m/z 625 resulted in a fragment with m/z 463, by loss of 162 amu corresponding to the loss of a
1 Data are expressed as means ± SD (n = 3). Values with the same letter in each column, are not significantly different (p > 0.05).
glucose moiety and m/z 301 corresponding to yet another glu- cose moiety. Fragmentation by MS3 of the aglycone obtained
2 Soluble extracts from onion skin and flesh were added to the fish
(m/z 301) originated fragments common to those obtained
at 0.1% level. Abbreviations are: MDA, malondialdehyde; and BHA,
from the fragmentation of quercetin (m/z 151, 179). Thus,
butylated hydroxyanisole.
from the respective fragmentation patterns we concluded that the peak at Rt 19.5 min corresponded to quercetin 3,40-
diglucoside. storage period. The samples arranged in the order of their
The high level of antioxidant activity in onions is
attrib- effectiveness in inhibiting the formation of TBARS (%) is as
uted to their flavonoid constituents, namely quercetin,
follows, red onion skin (68.46%) > pearl onion skin
kaempferol, myricetin, and catechin (Patil, Pike, & Yoo,
1995; (60.48%)>green shoot (53.29%)>BHA (51.89%)>red onion
Cook & Samman, 1996). Two major components
quercetin flesh (50.30%) > yellow onion skin (48.71%) > white onion skin
monoglucoside and quercetin diglucoside account for
80% (46.51%) > quercetin (36.72%) > Sprouted Red onion flesh
of the total flavonoids in onions (Bonaccorsi et al., 2008;
(9.78%) > Control. This trend is similar to that obtained in pre-
Rhodes & Price, 1996) with levels of quercetin glucosides
vious assays conducted in this study. Thus, onions are highly
much higher in onion than those in other vegetables
(Proteg- effective in inhibiting oxidation in a cooked meat system,
gente et al., 2002; Sellappan & Akoh, 2002; Shahidi &
Naczk, especially the extracts from the skin of red and pearl onions
2004). Similarly, Price and Rhodes (1997) reported that
querce- and green shoot, which were found to be better than BHA.
tin 3,40-O-glucoside and quercetin monoglucoside
(quercetin There was no significant difference between quercetin, white
40-O-glucoside) were the major flavonols in edible
portions skin, and green shoot on day 0 and day 7 (Table 3). Quercetin
of onions, however, they were mostly concentrated in the
alone showed a lower inhibition activity, probably because
skin. These conclusions are similar to the ones that can be
the onion extracts contained other antioxidant compounds,
drawn from the results in this study. the synergistic effects
of which improved its efficiency in
Additionally, in the present study, bound and esterified
inhibiting fish meat oxidation.
phenolic fractions of the onion samples were present in much lower concentrations as compared to the free form. None of 3.6.
HPLC analysis
the phenolic compounds could be detected in the esterified and bound fractions of the white onion peels, but quercetin The
identity of soluble and bound phenolic compounds were
and quercetin glucoside were detected at lower concentra-
ascertained using HPLC-DAD and HPLC–MS analyses and by
tions in the esterified or bound fractions of all other onion
comparison of their retention times and mass spectral data
peels. As expected, both the red onion and sprouted red
onion with those of the available standards and reports in the liter-
flesh had much lower concentration of phenolics as ature.
The predominant phenolic acids and flavonoids pres-
Table 4 – Content of prominent flavonoids (mg/g freeze dried sample) in the skin and flesh of four onion varieties quantified by
HPLC.
Sample Quercetin 3,40-diglucoside Quercetin Kaempferol
Free Esterified Bound Free Esterified Bound Free Esterified Bound
Pearl 9.59 2.54 1.44 8.33 0.03 0.23 1.36 0.01 0.01 Red skin 5.59 0.50 0.43 2.99 0.01 0.151 1.15 – 0.005 Yellow 2.57 0.16 0.19
3.11 0.007 0.01 1.13 – 0.006 White 0.004 – – 0.004 – – 0.003 – – Red flesh 2.54 0.05 0.01 0.15 – – 0.01 – – Red onion flesh,
sprouted 2.91 0.09 0.03 0.19 0.003 – 0.03 – – Green shoot (with chlorophyll) 2.32 0.06 0.01 0.14 – – 0.03 – – Green shoot
(dechlorophyllized) 2.11 0.08 0.02 0.11 0.001 – 0.02 – –
1199
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JOURNAL OF FUNCTIONAL FOODS
1200
JOURNAL OF FUNCTIONAL FOODS
5 (2013) 1191 –1203
DAD1 D, Sig=360,16 Ref=off (TALBISHI\TA3767A.D)
0 5 10 15 20 25 30
mAU
0
PS
RS
YS
2000
Quercetin
Quercetin
Quercetin
Kaempferol
1500
Quercetin 3,4 -́ diglucoside
Quercetin 3,4 -́ diglucoside
Quercetin 3,4 - diglucoside
1000
500
Kaempferol
Fig. 1 – HPLC chromatograms at 360 nm of free flavonoids extracted from skin of Pearl (PS) Red (RS) and Yellow (YS) onion
varieties.
0 5 10 15 20 25 30
mAU
DAD1 D, Sig=360,16 Ref=off (TALBISHI\TA3767C.D)
1200
1000
800
600
400
200
Kaempferol
0
mAU
DAD1 D, Sig=360,16 Ref=off (TALBISHI\TA3767D.D)
700
600
500
400
300
200
100
0
0 5 10 15 20 25
min
 compared to the skin. However, surprisingly, the sprouted red onion flesh was found to contain higher concentrations of both
quercetin and quercetin glucoside as compared to the ones not sprouted, probably due to reasons outlined in Section 3.1. As also
determined through the Folins total phen- olics test, pearl onion skin was found to contain the highest amount of total phenolic
acids, followed by red and yellow onions, sprouted red onion flesh, red onion flesh and white onion skin. This result is in
agreement with that of Patil et al. (1995) who reported that the red, pink, and yellow onions had higher amounts of quercetin than
white varieties. Prakash et al. (2007) also reported that the content of querce- tin decreased in all varieties from outer to inner
fleshy layers. Similarly, kaempferol was detected at much lower levels in
theesterifiedandboundformsofonionskinandflesh;thehigh- est amount observed in the free form of pearl onion skin fol- lowed by
red onion skin, yellow onion skin, red onion flesh, sprouted red onion flesh, green shoot of sprouted red onion flesh and white
onion skin. Sellappan and Akoh (2002) reported that the kaempferol in onions were found to be in minor quan- tities in
comparison to quercetin as kaempferol 3- and 4-gluco- sides. Onions grown in the United States were reported to have
kaempferol at 0.68 g/kg in the outer dry skin and 3–7 mg/kg in outer and inner skins of bulb (Bilyk, Cooper, & Sapers, 1984),
whereas onions grown in the United Kingdom did not have any detectable quantities of kaempferol (Crozier et al., 1997). These
variations may be due to many factors including variety, climatic conditions and maturity (Sellappan & Akoh, 2002).
Another set of experiments were carried out to test the efficiency of the solvent extraction method for dechlorophyl- lization
of green shoots (to reduce the interference of pro-oxi- dants) using spectrophotometry and HPLC data. This was done by
measuring the reduction in the absorbance of the sol- vent extracted sample at 660 nm (k
max
for chlorophyll). The efficiency of the extraction was confirmed using HPLC analy-
sis which showed the successful removal of chlorophyll (data not shown) without affecting the phenolic composition of the
extract (Table 4).
In Conclusion, these studies have demonstrated that onion skin, especially the darker (red and yellow) coloured ones are rich
in a number of antioxidant compounds that have a range of antioxidative properties. This study clearly established that onion skin
may serve as a promising source of natural antioxidants for the development of nutraceuticals or value-added products.
Moreover, the in vitro studies car- ried out further provided strong biochemical rationale for the benefit of onion-based diets and
the basis for further in vivo animal and human clinical studies. The important contribution of insoluble-bound phenolics in our
protocols further demonstrated that simple extraction of soluble phen- olics might lead to underestimation of phenolic contents
and their true efficacy in in vivo investigations.
Acknowledgements
We are grateful to King Saud University for partial support of this project. One of us (TA) thanks the Saudi Cultural Bureau for a
graduate scholarship.
JOURNAL OF FUNCTIONAL FOODS
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