Food Chemistry 133 (2012) 1653–1657

Analytical Methods Determination of flavonoid level variation in onion (Allium cepa L.)
infected by Fusarium oxysporum using liquid chromatography–tandem mass
spectrometry
Jung Han Lee a,1, Soo Jung Lee b,1, Semin Park a, Sung Woo Jeong a, Chi Yeon Kim c, Jong Sung Jin
d,*
,
Euh-Duck Jeong d, Youn-Sig Kwak e, Soo Taek Kim f, Dong Won Bae g, Gon-Sup Kim h, Sung Chul
Shin a,*
a Department of Chemistry, Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Republic of
Korea bDepartment of Food and Nutrition, Institute of Agriculture and Life Science, Gyeongsang National University, Jinju
660-701, Republic of Korea cDepartment of Dermatology, Institute of Health Science, Gyeongsang National University Hospital,
Jinju 660-702, Republic of Korea dDivision of High Technology Materials Research, Busan Center, Korea Basic Science
Institute (KBSI), Busan 618-230, Republic of Korea e Department of Applied Biology, Research Institute of Life Science,
Gyeongsang National University, Jinju 660-701, Republic of Korea fDepartment of Information and Statistics & Research
Institute of Natural Science, Gyeongsang National University, Jinju 660-701, Republic of Korea gCentral Laboratory,
Gyeongsang National University, Jinju 660-701, Republic of Korea h Research Institute of Life Science, College of Veterinary
Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea
articleinfo
Article history: Received 30 November 2011 Received in revised form 3 February 2012 Accepted 11 February 2012 Available
online 18 February 2012
Keywords: Allium cepa L. Fusarium basal rot Fusarium oxysporum Defense materials Flavonoid HPLC–MS/MS
abstract
In order to evaluate the flavonoid level variation in an onion (Allium cepa L.) infected by Fusarium oxysporum, the bulbs of a
healthy onion and of an infected one were analysed for flavonoids via high-per- formance liquid chromatography coupled with
tandem mass spectrometry (HPLC–MS/MS). Among ele- ven flavonoids characterised, isorhamnetin 40-O-galactoside (8) was
identified in an onion for the first time. When the healthy bulb was inoculated with the fungus, the two quercetin derivatives (4
and 7) and the two isorhamnetin derivatives (5 and 9) underwent concentration changes typical for the defense materials against
pathogens. The yellow granules that were accumulated on the abaxial epidermal cell layers after 8 days of inoculation were
confirmed as quercetin (10) and isorhamnetin (11). It was deduced that they were produced from flavonoids 4, 5, 7 and 9 by
hydrolysis enzyme of the fungus.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Onions (Allium cepa L.) are the oldest vegetable in continuous cultivation, dating back to at least 5000 BC. They can be
cultivated from the tropics to sub-polar regions (http://en.wikipedia.org/ wiki/Onion). As with other crops, onions are also
susceptible to the attack of numerous fungal pathogens that reduce yield and qual- ity. Fusarium basal rot (FBR) arising from the
invasion of the fungus Fusarium oxysporum through skin wounds is one of the most destructive soil-borne disease in onions
worldwide (Bayraktar, Türkkan, & Dolar, 2010). Once there is a wound in the onion tissue, the fungi can enter and begin to
spread up into the leaf system. Onions may become infected at any point in the field or during stor- age (Bayraktar et al., 2010;
Cramer, 2000). The disease begins with yellowing from the leaf tips, which is followed by progressive wilt- ing, curling and
brown discolouration on infected plants and the
symptoms move downward. In the advanced stage of infection, the pathogen causes pitting and decaying of the basal plate of
bulbs, watery rot and the development of white, fluffy mycelium (Sumner, 1995). Several cultural strategies including crop
rotation, pre-plant soil fumigation, avoiding wounds, storing bulbs in cool, biological control using fungal and bacterial
antagonists and growing varieties resistant against FBR have been proposed for controlling the patho- gen development. Among
the available options, the use of FBR- resistant varieties can effectively reduce losses to FBR. A number of intermediate and
long-day onion varieties are resistant against FBR infection (Cramer, 2000). However, little research has examined the chemical
and physiological resistance mechanisms of onions against FBR. This resistance may be attributed, at least in part, to the role of
the defense materials produced by the onion itself. Plants have a potency to activate a series of defense mechanisms to with-
stand the pathogen attack during infection (Chaves & Gianfagna, 2007). Therefore, understanding the defense materials and their
*
Corresponding authors. Tel.: +82 55 772 1484; fax: +82 55 772 1489 (S.C. Shin), tel.: +82 51 974 6114; fax: +82 51 974
6116 (J.S. Jin).
E-mail addresses: jinjs55@naver.com (J.S. Jin), sshin@gnu.ac.kr (S.C. Shin). 1 These authors contributed equally to this work.
functions by which the plant responds to pathogen attack may elucidate molecular markers for selecting genotypes with higher
disease resistance and reveal an alternative disease control strategy. Flavonoids with a basic skeleton of phenylbenzo-c-pyrone
are a
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2012.02.063
Contents lists available at SciVerse ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
member of the family of secondary metabolites ubiquitously found in the plants. Plant flavonoids have attracted great interest as
antiox- idants providing beneficial effects for human health. According to epidemiological reports, they have diverse biological
activities such as reducing the risk of various types of chronic disease (Erlund, 2004; Le Marchand, 2002), and exerting
antioxidant, anti-aging and antibacterial effects (Xu et al., 2007). They also play an important role in plants themselves as
physiologically-active components, including stress-protecting agent, attractants, feeding deterrents, signaling materials between
plants and soil microorganisms and defense materials against biotic and abiotic stresses (Harborne & Williams, 2000; Treutter,
2006; Vierheilig & Piché, 2002). Since several flavo- noids are toxic toward disease germs but not mammals, research interest
has focused on their possible application in subduing plant pathogens in an environmentally friendly manner (Benavente-Gar-
cia, Castillo, Marin, Ortuño, & Del Rio, 1997).
This work aimed at characterising the flavonoids of onion (Hanaro cultivar) infected by FBR using high-performance liquid
chromatography–tandem mass spectrometry (HPLC–MS/MS) and at elucidating the defense materials and molecular markers
against the disease. The flavonoid defense materials were profiled as the first step in understanding their physiological role in the
defense mechanism of onions and breeding onion varieties with stronger natural defenses against the FBR pathogen attack.
2. Materials and methods
2.1. Materials and chemicals
Onion (A. cepa L.) bulbs were harvested from Gyeongsangnam- do Agricultural Technology Education Center (GATEC),
Republic of Korea, in November 2010. The bulbs were authenticated as homo- zygous in genetic background by Dr. Chae-Shin
Lim of GATEC. The bulbs were lyophilised (PVTED50A, Ilsin Bio Base Co. Ltd., Yangju, Republic of Korea), and stored at
À70 °C until needed. Quercetin 3,40-O,O0-diglucoside, quercetin 40-O-glucoside and isorhamnetin were purchased from
Extrasynthese (Genay, France), and querce- tin, quercetin 3-O-glucoside and kaempferol from Sigma–Aldrich Co. (St. Louis,
MO, USA). The substances were used as standards for calibration after recrystallisation in ethanol. The purity of all standards
was confirmed by HPLC to be higher than 99%. All sol- vents and pure water were purchased from Duksan Pure Chemical Co.
Ltd. (Ansan, Republic of Korea). F. oxysporum (KACC No. 6076) was obtained from the Korean Agricultural Culture
Collection.
2.2. Pathogen inoculation
The fresh onion bulbs were sterilised with 70% ethanol by dip- ping for 10 s, washed with distilled water three times and
air-dried on a clean bench. F. oxysporum was sub-cultured on potato dex- trose agar (Difco, MD, USA) for 1 week at 27 ± 2 °C.
The bulbs were placed in rectangular plastic boxes that contained five layers of pa- per towel (Whatman Kimtowel L25) on the
bottom to maintain the humidity. The bulbs were wounded with a needle and inoculated with an agar disc containing mycelium
of F. oxysporum. The inocu- lated bulbs were incubated at 25 °C. The agar was removed from the diseased samples. The samples
were collected at one-day inter- vals for up to 9 days after inoculation. After liquid nitrogen was poured onto the samples, they
were ground with a pestle in a mor- tar, lyophilised and stored at À70 °C.
2.3. Extraction and purification of flavonoids
The lyophilised onion bulb (3 g) was poured into 70% methanol (50mL), homogenised using a Polytron blender (Brinkman
1654 J.H. Lee et al. / Food Chemistry 133 (2012) 1653–1657
Instruments, Westbury, NY, USA) for 2 min, and treated with a soni- cator (100 W, 42 kHz, Bransonics 3510RDTH, Danbury,
USA) for 10 min at room temperature. After repeated extraction (n = 3), the samples were combined and centrifuged for 10 min
at 3200g. The supernatant was transferred into a 200 mL flask. The solvent was re- moved under reduced pressure with a rotary
evaporator (Eyela NVC-2100, Tokyo Rikakikai Co. Ltd., Tokyo, Japan). The residue was dissolved in methanol/CH
2
Cl
2
(1:5, v/v, 2 mL) and loaded onto a homemade silica gel column (1 Â 5 cm, 70–230 mesh,
Merck 7734). The column was washed with hexane (5 mL) and eluted with ethyl acetate (20 mL). The solvent was removed
under reduced pressure. The residue was reconstituted in methanol (1 mL), filtered through 0.45 lm cellulose membrane,
transferred into glass vials and stored at À20 °C until analysis.
2.4. HPLC–MS/MS
HPLC–MS/MS experiments were performed according to the lit- erature method (Kim et al., 2011) except for the use of a
solvent system consisting of 0.1% aqueous acetic acid (A) and methanol (B). The gradient conditions of the mobile phase were
from 20% to 30% of B over 10 min, increased to 70% B over 15 min, decreased to 20% B over 5 min, and then isocratic elution
for 10 min.
2.5. Statistical analysis
All determinations were performed in quintuplicate and the mean ± standard deviation values were calculated. The data were
subjected to analysis of variance for repeated measurement (SAS version 9.1.3) and significant differences were between means
(p < 0.001).
3. Results and discussion
3.1. Separation and characterisation
The flavonoid mixture was isolated from the onion bulbs by extraction with 70% aqueous methanol. Extraction with aqueous
alcoholic solvent followed by isolation by reverse phase chroma- tography over C
18
column has been extensively used for purifica- tion of plant flavonoids (Dou, Lee, Tzen, & Lee, 2008). In this
work, the aqueous methanol extract was chromatographed over silica gel using a mixture of methanol and CH
2
Cl
2
as an eluent. Silica gel is a cheaper absorbent than C
18
. It has been demonstrated that flavonoid mixture can be effectively isolated from the plant
ex- tract by chromatography over silica gel (Lee et al., 2011). The iso- lated flavonoids were characterised by reverse phase
HPLC (RP- HPLC), MS/MS in negative ion mode and compared with reported data. Since flavonoid compounds are moderately
polar, they are usually analysed primarily on RP-HPLC coupled to ESI-MS (Choi et al., 2010). After testing several RP-columns
for their suitability, a Zorbax SB-C
18
column was chosen because it provides good peak separation and symmetry. The binary solvent system of
0.1% aqueous acetic acid and methanol was used as the mobile phase because it provided good separation between the chro-
matographic peaks. A mass spectrometer with electrospray inter- face was operated over the scan range m/z 100–1000 in
negative ion mode.
The eleven flavonoids (1–11) are labeled between 10 to 60 min in the chromatogram of the healthy and diseased onion bulbs
re- corded at 360 nm at 8 days after inoculation (Fig. 1). The structures and HPLC–MS/MS data of the eleven flavonoids are
shown in Table 1. Ten flavonoids (1–7, 9–11) have previously been characterised for other onion varieties (Bonaccorsi, Caristi,
Gargiulli, & Leuzzi, 2005; Grzelak, Milala, Król, Adamicki, & Badełek, 2009; Lee &
Fig. 1. HPLC chromatograms of the flavonoids isolated from the healthy onion bulb (A) and from that infected by Fusarium
oxysporum 8 days post-infection (B). (1) quercetin 3,7,40-O,O0,O00-triglucoside; (2) quercetin 3,40-O,O0-diglcoside; (3)
quercetin 7,40-O,O0-diglucoside; (4) quercetin 3,40-O,O0-diglucoside; (5) isorhamnetin 3,40-O,O0-diglucoside; (6) quercetin
3-O-glucoside; (7) quercetin 40-glucoside; (8) isorhamnetin 40-galactoside; (9) isorhamnetin 40-glucoside; (10) quercetin; (11)
isorhamnetin. Detection wavelength: 360 nm.
Table 1 Structures and mass spectral data of the 11 flavonoids of healthy and diseased onion bulb.
r.t [MÀH]À MS/MS Compounds
13.1 787 625463301 Quercetin 3,7,40-O,O0,O00-triglucoside (1); R
1
=R
2
=R
4
= Glu, R
3
=
H 18.2 625 625463301 Quercetin 3,40-O,O0-diglcoside (2); R
1
+R
2
+R
3
+R
4
=
2Glu + 2H 22.8 625 625463301 Quercetin 7,40-O,O0-diglucoside (3); R
1
=R
2
= H, R
2
=R
4
=
Glu, 26.9 625 625463301 Quercetin 3,40-O,O0-diglucoside (4); R
1
=R
2
= Glu, R
3
=R
4
=
H 28.1 639 639477313 Isorhamnetin 3,40-O,O0-diglucoside (5); R
1
=R
2
= Glu, R
3
= CH
3
33.7 463 463301 Quercetin 3-O-glucoside (6); R
1
= Glu, R
2
=R
3
=R
4
=H
36.2 463 463 301 178 151 Quercetin 40-O-glucoside (7); R
1
=R
3
=R
4
= H, R
2
= Glu
37.3 477 477315 Isorhamnetin 40-O-galactoside (8); R
1
=R
3
=R
4
= H, R
2
= Gal, R
3
= CH
3
38.6 477 477315 Isorhamnetin 40-O-glucoside (9); R
1
=R
3
=R
4
= H, R
2
= Glu, R
3
= CH
3
43.8 301 179151 Quercetin (10); R
1
=R
2
=R
3
=R
4
= H 51.8 315 315
301 271 151 107 Isorhamnetin (11); R
1
=R
2
=R
4
= H, R
3
= CH
3
r.t: retention time (min); Glu: glucose; Gal: galactose; Data were obtained at negative ion mode.
Mitchell, 2011). Flavonoid (8) was identified tentatively for the first time for the onion by mass fragmentation patterns and com-
parison with the literature data (Parejo et al., 2004). The MS/MS of flavonoid 8 produced [MÀH]À at m/z 477, which fragmented
into m/z 315 ([MÀH]À–galactosyl), corresponding to the isorhamnetin unit. Since flavonoid grafted to the galactose unit elutes
slightly more quickly than that grafted by a glucose in a C
18
RP column (Choi et al., 2010), the shorter retention time (r.t. = 37.3) of
flavo- noid 8 compared to that of isorhamnetin 40-O-glucoside (9, r.t. = 38.6), which is previously known for other onion
varieties, al- lowed it to be assigned as isorhamnetin 40-O-galactoside.
Table 2 Validation data for five representative standards. 3.2. Validation
Standards Recovery ± RSD (%) (n = 5) r2 LOD (mg/L) LOQ (mg/L)
All of the flavonoids were quantified using chromatograms ex-
4 6 88.1 ± 2.9 82.3 ± 2.2 0.999 0.037 0.075 0.998 0.021 0.042 tracted at 360nm. The quantification method was validated in
7 84.6 ± 1.8 0.999 0.009 0.018 terms of specificity,
linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision, according to the
10 11 86.0 ± 9.6 87.6 ± 3.5 0.997 0.017 0.035 0.998 0.025 0.051
guidelines of the International Conference of Harmonisation
RSD: relative standard deviation; r2: correlation
coefficients; LOD: limit of detec- (ICH).The validation data are compiled in Table 2.
tion; LOQ: limit of quantitation.
J.H. Lee et al. / Food Chemistry 133 (2012) 1653–1657 1655
3.2.1. Specificity
The specificity is the non-interference with other substances detected in the region of interest of the analytic method. As
shown in Fig. 1, the analyte peaks on the chromatogram were well sepa- rated without any other peak interfering, indicating the
good specificity.
3.2.2. Linearity
Quercetin 3,40-O,O0-diglcoside (4), quercetin 3-O-glucoside (6), quercetin 40-O-glucoside (7), quercetin (10) and
isorhamnetin
(11) were quantified using the linear calibration curves of the indi- vidual corresponding standards. The quercetin derivatives
(1–3) were quantified as their corresponding aglycone, 10, and isorham- netin derivatives (5, 8, 9) as aglycone, 11. Plant
flavonoids have been routinely quantified using a standard curve of structurally re- lated compounds (McGhie, Hunt, & Barnett,
2005). The calibration curves were constructed from the peak area ratios of analyte/IS (kaempferol) versus analyte/IS
concentrations using a 1/x (x: con- centration) weighted linear regression (n = 5). The concentrations of standards spanned eight
points 0.1, 1, 5, 10, 50, 100, 200 and 500 mg/L. The IS concentration was 50 mg/l. The regression equa- tion was estimated in the
form of y = ax + b, where y and x repre- sent the peak area and the concentration of each compound, respectively. Regression
analysis presented correlation coefficients (r2) > 0.997 for the compounds (n = 5), indicating a good linearity.
3.2.3. LOD and LOQ
The performance limit of the chemical assay was represented by LOD and LOQ, which were estimated experimentally by
injecting individual standard solutions to HPLC until the signal-to-noise ra- tio for the standards reached ratios of 3:1 and 10:1,
respectively. The results demonstrated that the developed method was very sensitive with LODs ranging from 0.009 to 0.037
mg/L, and LOQs from 0.018 to 0.075 mg/L.
3.2.4. Accuracy and precision
The accuracy of the methods was evaluated as recovery = A/IS-C/ B/IS-C, where A is the peak area obtained for the analyte
spiked pre-extraction, B that obtained for the analyte spiked post- extraction and C that of blank extraction. The experiment was
car- ried out in quintuplicate at a concentration of 50 mg/L and an IS concentration of 50 mg/L. The measured recoveries were in
the range of 82.3–88.1%. The precision of the method represented in terms of RSD fell within the range of 1.8–9.6%. The
determined val- ues of accuracy and precision were both acceptable.
3.3. Monitoring of flavonoid defense materials in onion bulb infected by F. oxysporum
The validated method was applied to the simultaneous quanti- fication of the eleven flavonoids. The contents of individual
flavo- noids in the healthy control bulb of the onion are listed in Table 3. The total amount of the flavonoids identified in the
health bulbs was 137.1±0.067mg/kg fresh bulb weight. Among the eleven flavonoids, the concentration of component 4 was the
highest and 6 the lowest. Quercetin 10 and isorhamnetin 11 were not detected.
1656 J.H. Lee et al. / Food Chemistry 133 (2012) 1653–1657
To understand the role of flavonoids as defense materials in the onion against the fungus F. oxysporum, the healthy bulb was
inoc- ulated with F. oxysporum. Observable macroscopic disease symp- toms started to develop as water-soaked lesions 3days
after inoculation, and the fungal hyphae grew out of the inoculation site with white to pink colour 5 days after inoculation (Fig.
2A). De- cayed tissues became sunken and turned grayish brown, and dis- ease lesions were increased in size after 8days of
inoculation (Fig. 2B). Interestingly, a strong yellowish colour was observed at the edge of the disease lesions on the abaxial
epidermal cell layers after 8 days of inoculation. A dissection microscope image (Â50, Nikon SMZ800, Tokyo, Japan) showed
the accumulation of numer- ous yellow granules within the abaxial epidermal cell wall (Fig. 2C).
When the healthy onion bulbs were inoculated with F. oxyspo- rum, among the eleven flavonoids determined, the
concentrations of the two quercetin derivatives (4 and 7) and the two isorhamnetin derivatives (5 and 9) underwent substantial
and statistically signif- icant changes (p < 0.001). The contents of the flavonoids 4, 5, 7 and 9 were monitored by HPLC over the
9-day post-inoculation period and the results are shown in Fig. 3. At the early stage of infection, the concentration of flavonoid 4
decreased gradually during 3 days and that of 5, 7 and 9 during 4 days. At the middle stage, the concen- trations of flavonoids 4,
5, 7 and 9 increased progressively to a peak concentration 6 days after infection. The concentrations of flavo- noids 4, 5, 7 and 9
then decreased progressively after the peak concentration. This type of concentration change may be closely associated with the
infection process and has been reported as char- acteristic of the defense materials (Jasin ́ ski, Kachlicki, Rodziewicz,
Figlerowicz, & Stobiecki, 2009; Kim et al., 2011). The pathological response of the defense materials against pathogen infection
typi- cally follows a three-step process. (1) Pre-consumption of the con- stitutively prepared defense materials before the
initiation of their biosynthesis in response to the pathogen attack. (2) The biosynthe- sis of the defense materials by the plant and
the overwhelming of the defense function against the pathogen metabolism. (3) The col-
Table 3 Contents of the nine flavonoids detected in the healthy onion bulb (mg/kg).a
Compounds Meana ± SD
1 5.3 ± 0.003 2 5.4 ± 0.005 3 13.0 ± 0.006 4 73.0 ± 0.080 5 4.9 ± 0.006 6 0.7 ± 0.000 7 20.3 ± 0.007 8 1.6 ± 0.004 9 12.9 ± 0.008
Total 137.1 ± 0.067
a Data are the mean±SD (stan- dard deviation) of triplicate deter- minations by HPLC–UV method; SD: standard deviation.
Fig. 2. (A) Image of healthy bulb; (B) image 5 days after inoculation; (C) image 8days after inoculation; (D) dissection
microscope image (Â50) 8days after inoculation, yellow granules accumulated within the abaxial epidermal cell wall are seen;
(E) image after enucleated with a needle using a dissection microscope; (F) LC and MS of enucleated granules. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
lapse of the competition balance and defeat of the defense mecha- nism of the plant. Few reports have been published on the anti-
pathogenic activity of the isorhamnetin and quercetin derivatives. Isorhamnetin (11) showed anti-pathogenic activity against
Verticil- lium albo-atrum (Picman, Schneider, & Picman, 1995). Quercetin derivatives inhibited the growth of blue mould in
pome fruits caused by Penicillium expansum (Sanzani, Schena, Nigro, de Girol- amo, & Ippolito, 2009) and conidial germination
of Neurospora cras- sa (Lattanzio, Lattanzio, & Cardinali, 2006).
The disease progress is depicted in Fig. 2; A: image of healthy bulb, B: image 5 days after inoculation, C: image 8 days after
inoc- ulation. The accumulated yellow granules were enucleated with a tapering needle using a dissection microscope (Â50) (Fig.
2D and E). The retention times on chromatogram and MS of the enucleated granules were in accordance of those of flavonoids
10 and 11 (Fig. 2F). The abrupt accumulation of flavonoids 10 and 11 observed 8 days post-inoculation was attributed to the
hydrolysis of the indi- vidual glycosidic bonds of flavonoid glycosides 4, 5, 7 and 9 by the hydrolysing enzyme produced by F.
oxysporum. The conversion of the flavanone glycosides to flavanone aglycones by the hydrolysing enzyme of fungus was
recently reported (Kim et al., 2011). While flavonoids 4, 5, 7 and 9 are highly soluble to water, flavonoids 10 and 11 should be
less soluble due to the absence of a glycosidic moi- ety. 10 and 11 were crystallised within the cell immediately after their
production due to their low solubility.
4. Conclusions
HPLC–MS/MS has been proved suitable for evaluating the flavo- noid concentration variation in an onion (A. cepa L.)
infected by F. oxysporum. The two quercetin derivatives (4 and 7) and the two isorhamnetin derivatives (5 and 9) showed
concentration changes typical for the defense materials against pathogens. At the final stage of the disease development, the
defense materials were hydrolysed to their aglycones, quercetin (10) and isorhamnetin (11) by the hydrolysis enzyme of the
fungus.
Acknowledgement
This work was supported by a KBSI Grant (T32710) to S.C. Shin and J.S. Jin.
J.H. Lee et al. / Food Chemistry 133 (2012) 1653–1657 1657
Fig. 3. Content change of flavonoids in the healthy and diseased onion bulb from 0 (control) to 9 days after inoculation (p <
0.001). Data are given as mean ± SD, n = 5.
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