Open Journal of Applied Sciences, 2012, 2, 209-215
doi:10.4236/ojapps.2012.24031 Published Online December 2012 (
The Preparation of Preserved Shallot Powders and a Pilot
Study of the Antioxidative Effect of Their Aqueous Extracts
on the Formation of Hydroxyl Radical Species
Ji-Yuan Liang*, An-Chi Hsu*, Xin-Yu Lan*, Kuan-Yu Chen, Po-Shuan Chen, Wei-Ming Chou,
Kuei-Yan Liou, Dung-Yu Peng, Jeu-Ming P. Yuann#
Department of Biotechnology, Ming Chuan University, Taoyuan County, Taiwan
Received August 30, 2012; revised September 29, 2012; accepted October 10, 2012
In order to preserve the nutrients in shallots, after harvest, various protocols, including incubation, drying or lyophiliza-
tion of the shallot are developed in this study. Using aqueous extracts of ground shallot powders, this study examines
the antioxidative properties of shallots on the formation of hydroxyl radical species (OH) generated via a Fenton-type
reaction. A ribose degradation assay shows that all aqueous extracts of shallot prepared in this study exhibit enhanced
levels of OH, suggesting that processed shallot, like strong reductants such as ascorbate, has a strong reducing power,
which converts Fe3+ to Fe2+ in a Fenton’s reaction and increases the levels of OH. A DNA integrity assay shows that
fragmentation of super-coiled plasmid DNA, pGEM-7Zf(-), by OH is diminished in the presence of all shallot aqueous
extracts, albeit to various extents. Finally, electron paramagnetic resonance (EPR) experiments show that lyophilized
shallot completely scavenges OH, as evidenced by the disappearance of the EPR-active reaction product generated be-
tween spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and OH. The results of this study show the potential of a
daily intake of preserved shallot to boost antioxidative protection against the toxicity of OH or any other damaging
Keywords: 2’-Deoxy-D-Ribose; EPR; Fenton’s Reaction; Hydroxyl Radical; Plasmid DNA; Shallot
1. Introduction
Extracts of plant foods have been shown to exhibit an
antioxidant effect and a capacity to scavenge free radi-
cals, to protect against external and endogenous agents in
the treatment of human health problems [1-4]. Of these
extracts, shallot (Allium ascalonicum L.) has been used
as a food additive in Chinese society for generations.
Shallot is mainly grown in southern Taiwan, but it can be
harvested only once a year. Therefore, the preservation
of shallot is an important issue for both farmers and peo-
ple who use shallot as a dietary additive, because of its
unique fragrance. In Taiwan, shallot is mostly preserved
by frying, which maintains its fragrance, but this may not
be a good method of preserving its nutrients, since the
consumption of deep-fried food has been identified as one
of the risk factors of lung cancer, in Sichuan, China [5].
This study develops three different protocols for re-
moving the water in shallot purchased from a local food
store. Firstly, the shallot is incubated to increase its fla-
vor, presumably through a Maillard reaction, followed by
heating. Secondly, the shallot is directly dried by heating,
in order to ascertain whether incubation has any effect on
the antioxidative activity of the incubated shallot. Thirdly,
the shallot is firstly frozen, followed by lyophilization, in
order to develop a protocol by which shallot can be pre-
served at lower temperature. The dried shallot prepared
according to these methods is then ground into a powder,
for long-term storage.
Analysis of the shallot extracts has confirmed the pre-
sence of flavone and polyphenolic compounds, such as
quercetin and its derivatives, which suggest that shallot
may exhibit antioxidant properties [6-8]. In order to ex-
amine the antioxidative properties of shallot prepared
according to these proposed protocols, aqueous extracts
of the three types of shallot powder were subjected to
reactions, in order to investigate their ability to scavenge
OH generated via a Fenton-type reaction (Fe2+ + H2O2
Fe3+ + OH + OH). Hydroxyl radical, an oxy-
gen-derived radical species, has been implicated in the
carcinogenesis of many types of cancer, such as gastric
cancer, by causing tissue damage and thus allowing the
*These three authors contributed equally to this work.
#Corresponding author.
Copyright © 2012 SciRes. OJAppS
cancer to spread [9]. The acceleration of gastric carcino-
genesis by sodium chloride may be mediated by OH [10].
Moreover, in a meta-analysis, consumption of large
amounts of allium vegetables (onions, garlic, shallot) has
recently been shown to reduce the risk of gastric cancer
[11], but there is no credible evidence to support a rela-
tionship between garlic intake and a reduced risk of gas-
tric, breast, lung or endometrial cancer [12]. Extracts of
shallot have previously been shown to scavenge posi-
tively charged radical species, 2,2’-azinobis(3-ethylben-
zothiazoline-6-sulfonic acid), ABTS•+, more efficiently
than those of garlic, although shallot and garlic similarly
inhibit the protein hydroperoxides caused by OH [13].
The hydroxyl radical is more easily diffused and more
toxic than ABTS•+ [14], but a comparison of the direct
OH-scavenging ability of shallot and garlic aqueous ex-
tracts has not been documented. In this study, the OH-
scavenging effects of shallot were evaluated by their in-
fluence on ribose degradation, super-coiled plasmid DNA
integration and EPR spin-trapping. The results show that
aqueous extracts of shallot powders behave as a reduc-
tant in a ribose degradation assay, while in the DNA in-
tegrity assay and EPR experiments, they act as a OH
scavenger. Because of the widespread use of shallot and
other types of allium vegetables by many populations,
this study provides important nutritional guidelines for
the use of shallot as a food additive.
2. Materials and Methods
All chemicals were reagent grade or better and used
without further purification. The de-ionized water (D.I.
H2O) used to make solutions was redistilled using a Wa-
ters water distillation system. The fresh shallot and garlic
used for the EPR experiments were purchased from a
local food store in Taoyuan County, Taiwan, and pre-
served immediately upon arrival, by freezing at –20˚C,
until preparation commenced. Ferrous ammonium sulfate
6-hydrate [Fe(NH4)2(SO4)2·6H2O] was purchased from
J.T. Baker (Phillipsburg, NJ). Ascorbic acid, 2’-deoxy-
D-ribose, DMPO, 2’-thiobarbituric acid (TBA) and tris
(hydroxymethyl)aminomethane (Trizma base or Tris) were
purchased from Sigma-Aldrich (St. Louis, MO). Hydro-
gen peroxide (H2O2) was purchased from T.K. Chemical
Co. (Tainan, Taiwan). The reagent used to stain DNA
was HealthView Nucleic Acid Stain, purchased from Ge-
nomics Co. (Shiji, Taiwan).
All solutions were prepared using D.I. water or the
solvents noted; otherwise. Stock solutions of ferrous
ammonium sulfate (6 mM) and 2’-deoxy-D-ribose (1 mM
in 60 mM Tris at pH 7.0) were prepared, loaded in 1.5 ml
Eppendorf tubes and frozen at –20˚C, until experimenta-
tion. Because of their instability, shallot aqueous extracts,
H2O2 (60 mM) and TBA (1% in 50 mM NaOH) were all
freshly prepared, prior to each experiment.
2.1. Preparation of Preserved Shallot
This study develops three different protocols to preserve
shallot, as described briefly below. Firstly, the shallot
was incubated in an incubator, at a constant 85˚C and
85% humidity, for 12 days, followed by three-days of
heat drying in an oven at 60˚C. The heat-dried shallot,
shallot was directly dried in an oven at 60˚C for three
days. The lyophilized shallot was frozen and lyophilized,
to remove all water content. After preparation, all three
types of shallot were ground to a powder, for anaerobic
storage. The shallot powders were freshly dissolved in
D.I. water, to produce a 10% (w/v) aqueous extract, fol-
lowed by centrifugation at 10,000 g, to remove debris
that was not dissolved, and the supernatant was saved for
2.2. Assay of 2’-deoxy-D-ribose Degradation
The generation of OH via a Fenton-type reaction was
quantified using 2’-deoxy-D-ribose oxidative degradation,
as described previously, with minor modifications [15].
The assay quantifies the 2’-deoxy-D-ribose degradation
product, malondialdehyde (MDA), by its condensation
with TBA under acidic conditions. In a typical experi-
ment, 30 μl 2’-deoxy-D-ribose (1 mM in 60 mM Tris at
pH 7.0) was first added to the bottom of a 1.5-ml Ep-
pendorf tube. Then, solutions of H2O2 (60 mM) and D.I.
water (control), or an aqueous solution of shallot extract
in a volume of 10 μl each were added as two individual
drops to the wall of the tube. Each reaction was initiated
by firstly mixing the two solution drops on the wall with
a pipette tip containing 10 μl Fe2+ (6 mM in 60 mM Tris
at pH 7.0) and then introducing the mixed solution to the
2’-deoxy-D-ribose solution, followed by the addition of
Fe2+ to the reaction solution. Therefore, the final volume
of each reaction solution was 60 μl and the concentra-
tions of 2’-deoxy-D-ribose, H2O2, and Fe2+ were 0.5, 10,
and 1 mM, respectively. After 80 s (1.33 min), the reac-
tions were terminated by adding 4% phosphoric acid (v/v)
of 60 μl, followed by 60 μl of 1% TBA. After boiling for
15 min, 60 mM Tris at pH 7.0 of 720 μl was then added
to each solution, to produce a final volume of 900 μl,
prior to spectrophotometric measurement at 532 nm. The
measurement of the absorbance of each reaction solution
was performed at room temperature in a Perkin-Elmer
spectrophotometer of type Lambda35, equipped with a
quartz cuvette of 1 cm light path. In order to remove the
absorbance from the shallot aqueous solution in each
experiment, the absorbance from a solution processed
under the same conditions, in the absence of a Fenton’s
reaction, was measured and subtracted from the total
Copyright © 2012 SciRes. OJAppS
J.-Y. LIANG ET AL. 211
2.3. DNA Integrity Assay
The DNA integrity assay was performed using super-
coiled plasmid DNA as the target molecule. Plasmid
DNA, pGEM-7Zf(-), was transformed into E. coli, DH5α,
and grown overnight in LB broth at 37˚C. The culture
was then harvested and the DNA was purified using a
Plasmid Miniprep kit (BioKit, Miaoli, Taiwan). After
purification, 6 μl plasmid DNA dissolved in D.I. water
was added drop-wise onto a piece of parafilm prior to the
addition of other reagents. Then, 2 μl D.I. water or shal-
lot aqueous extract and 2 μl 60 mM H2O2 were added
onto the parafilm close to the plasmid DNA solution as
two drops. Finally, a pipette tip holding 6 mM Fe2+ of 2
μl in 60 mM Tris at pH 7.0 was used to mix the water or
shallot aqueous extract and H2O2 together and to transfer
this mixed solution into the DNA solution, followed by
the addition of Fe2+, to initiate the reaction. After 80 s,
the reaction was quenched by adding 2 μl loading dye
(0.25% bromophenol blue and 40% sucrose), prior to
electrophoresis in a 1.5% agarose gel. The plasmid DNA
was visualized by internally staining the gel with Health
View Nucleic Acid Stain.
2.4. EPR Experiments
Previous results showed that DMPO can be oxidized to
generate the EPR-active species, DMPO-OH, under
acidic conditions (pH 3.0 - 6.0), without involving OH
(data not shown). The EPR experiments in this study
were therefore performed under slightly alkaline condi-
tions (pH 8.0) to avoid this situation. Stock DMPO solu-
tion of 200 mM was produced by dissolving DMPO in
Tris-HCl (100 mM at pH 8.0), followed by washing with
charcoal, to remove impurities, and filtering through an
acrodisc of 0.45 μm. In a typical EPR experiment, 45 μl
DMPO solution was first added onto a piece of parafilm.
Fifteen-μl D.I. water or 10% shallot aqueous extract and
15 μl H2O2 were added as two drops, close to the DMPO
solution. Then, a pipette tip holding 15 μl Fe2+ in 100
mM Tris-HCl at pH 8.0 was used to mix water or shallot
aqueous extracts and H2O2 together and to transfer the
mixed solution to the DMPO solution, followed by the
injection of Fe2+ solution. Then, the entire reaction solu-
tion was drawn into a capillary tube, sealed with Vase-
line grease and loaded into an EPR quartz tube for meas-
urement. The reaction was allowed to continue for 8 min,
prior to EPR data collection. A Bruker EMX-10 X-band
EPR spectrometer was used for the measurement of
DMPO-OH at room temperature. The parameters for
obtaining the EPR spectra were as follows. Instrumental
conditions were: microwave frequency, 9.765 GHz; mi-
crowave power, 19.97 mW; modulation amplitude, 1G;
scan time, 168 s; time constant, 20.48 ms and scan range,
100 G. The EPR measurements for DMPO alone were
performed before and after the experiment, to ensure that
the EPR-active species produced by the oxidation of
DMPO was not interfering with the experiments. The
results show that no EPR signals from these species were
2.5. Statistical Analysis
In the ribose degradation experiments, the results are
expressed as mean ± standard deviation (SD). A one-way
analysis of variance (ANOVA) was performed to com-
pare means of two or more samples. When statistically
significant differences were found, an unpaired Student’s
t-test was used. A value of p < 0.05 is considered to be
statistically significant.
3. Results
3.1. Preparations of Preserved Shallot
This study develops three different protocols to preserve
shallot in powder form, as described above. It appears
that heat-dried shallot, with or without incubation, be-
comes dark, while lyophilized shallot remains pink, with
little change in its color. Additionally, all three types of
shallot were ground into powder, for long-term storage,
without much difficulty. The antioxidative experiments
conducted in this study show that the dried powder form
appears to be effective in preserving the nutrient content
of shallot or other allium vegetables.
3.2. 2’-Dexoy- D-Ribose Degradation Assay
In order to examine the effect of shallot aqueous extract
on ribose degradation due to hydrogen abstraction caused
by OH, 2’-dexoy-D-ribose was subjected to a Fen-
ton-type reaction, to generate reactive OH, and one of
the rearranged products, malondialdehyde (MDA) from
ribose degradation, and its complex with TBA, MDA-
TBA, were measured in acidic conditions at 532 nm. As
shown in Figure 1, the level of the MDA-TBA complex
is relatively low in the absence of any shallot aqueous
extracts, whereas the levels of the complex are greatly
increased in the presence of aqueous extracts of all three
types of shallot prepared in this study. In other words, in
the ribose degradation assay, the aqueous extracts of
shallot prepared under current conditions do not exhibit
any OH-scavenging effects against OH, but instead in-
crease the degradation of ribose, as shown in Figure 1.
The increase in ribose degradation in the presence of all
three types of shallot aqueous extract is most likely due
to the reducing power of shallot, which converts Fe3+ to
Fe2+, which then reacts with excess H2O2 (60 mM) to
generate OH continuously. Under the same conditions,
Copyright © 2012 SciRes. OJAppS
Control A B C
Figure 1. Ribose degradation by hydroxyl radical species
generated via a Fenton’s reaction, in the presence of aque-
ous extracts of (A) Incubated shallot; (B) Heat-dried shallot
and (C) Lyophilized shallot. The control used D.I. water.
Data are represented as mean ± SD, where n = 3.
ascorbate, a reductant known to enhance a Fenton-type
reaction [16], causes a much greater increase in the levels
of MDA-TBA (data not shown).
3.3. DNA Integrity Assay
DNA cleavage caused by OH have been extensively
studied [17]. In order to examine the levels of DNA
strand breaks caused by OH in the presence of shallot
aqueous extracts, super-coiled plasmid DNA was al-
lowed to react with OH generated via a Fenton-type re-
action, in the absence or presence of shallot aqueous ex-
tracts. In Figure 2, lane 1, super-coiled plasmid DNA
alone is visualized. In lane 2, complete DNA digestion
caused by OH is seen, as shown by the disappearance of
DNA bands in gel analysis. In lanes 3-5, in the presence
of 10% aqueous extracts of shallot, plasmid DNA bands
are all retained, although mostly in linear form, instead of
super-coiled forms, with the heat-dried shallot aqueous
extract retaining the largest amount of both super-coiled
and linear DNA. These results suggest that the ingredi-
ents in the aqueous extracts of shallot scavenge OH and
decrease the levels of DNA cleavage.
3.4. EPR Experiments
The results from ribose degradation and DNA integrity
assay seem contradictory, so the OH scavenging effect
of the aqueous extract of lyophilized shallot on the levels
of OH were further examined using EPR and spin trap,
DMPO. Hydroxyl radical reacts with DMPO to generate
EPR-active nitroxide, DMPO-OH, as evidenced by a
quartet EPR splitting of 1:2:2:1 ratio [18]. Lyophilized
shallot was selected for the EPR experiment, mainly be-
cause of its ease of preparation compared to the other
two types of shallot prepared in this study. For compari-
son purposes, lyophilized garlic aqueous extract was
used as a control, since garlic (both the homogenate of
10% physiological saline solution and its supernatant)
has been shown to decrease the level of OH trapped by
phenyl-butyl-nitrone for EPR measurements [19]. As
shown in Figure 3(a), OH generated in a Fenton-type
reaction reacts with DMPO to generate the EPR-active
species, DMPO-OH. In Figure 3(b), under the same con-
ditions as in 3(a), except in the presence of lyophilized
shallot aqueous extract, it is seen that the EPR signals of
DMPO-OH completely disappear. In Figure 3(c), under
the same conditions as 3(a), except in the presence of ly-
ophilized garlic aqueous extract, the EPR signals of
M 1 2 3 4 5
Figure 2. The DNA integrity assay of the aqueous extracts
of shallot powders prepared in this study: Lane M, 1 kb
DNA marker; Lane 1, plasmid DNA, pGEM-7Zf(-), alone;
Lane 2, plasmid DNA in a Fenton’s reaction; Lanes 3-5,
plasmid DNA in a Fenton’s reaction in the presence of
aqueous extracts of incubated, heat-dried and lyophilized
shallot powders, respectively. The notations, -oc, -l and -sc,
represent open-circular, linear and super-coiled plasmid
DNA, respectively.
3425 3450 3475 3500 3525
Magnetic field (Gauss)
Figure 3. The EPR spectra of DMPO-OH generated in a
Fenton’s reaction in the presence of (a) D.I. water; (b)
Aqueous extract of lyophilized shallot powder and (c) Aque-
ous extract of lyophilized garlic powder.
Copyright © 2012 SciRes. OJAppS
J.-Y. LIANG ET AL. 213
DMPO-OH decrease to a much lower level than that of
the control, but not to the extent that occurs in the pres-
ence of lyophilized shallot aqueous extract.
4. Discussion
The results of this study show that shallots harvested in
Taiwan can be preserved in powder form for long-term
storage without much difficulty, using three different
protocols. The findings for the antioxidative effects of
shallot aqueous extracts reveal that in the ribose degrada-
tion assay all three shallot aqueous extracts appear to
increase the amount of the OH radical, instead of scav-
enging, as expected. In the DNA integrity assay, shallot
aqueous extracts are seen to scavenge OH and to de-
crease the level of DNA cleavage. Lastly, in the EPR
experiments, lyophilized shallot aqueous extract is seen
to have a scavenging ability and completely eliminates
OH, compared with garlic aqueous extract, a known OH
scavenger [19], which only partially eliminates OH.
Meta-analysis has shown that shallot reduces the risk
of gastric cancer, but only if a large amount of shallot is
consumed [11]. The solubility and bioavailability of ex-
emestane, an irreversible aromatase inhibitor, has been
found to greatly improve in powder and tablet form [20],
so the beneficial effects of shallot powders prepared us-
ing these protocols are increased if the shallot powder is
dissolved in water or other types of solvents. The long-
term health benefits of shallot aqueous extracts due to
their antioxidative effect on human diseases deserves
further investigation.
In the ribose degradation assay, all aqueous extracts of
shallot powders increase the amount of OH, similarly to
ascorbate, by reducing Fe3+ to Fe2+ in a Fenton’s reaction,
to continuously increase the levels of OH. This can be
toxic, since an ascorbate-driven Fenton’s reaction may be
an important mechanism for cell death in biological sys-
tems [16]. However, in the DNA integrity assay, all aqu-
eous extracts of shallot powders demonstrate OH-scav-
enging by decreasing the levels of DNA cleavage caused
by OH. The contradiction in the results from these two
assays can be partially explained by the hydrogen acces-
sibility of ribose rings on the 2’-deoxy-D-ribose in ribose
degradation assay and that of the sugar rings of duplex
DNA in the DNA integrity assay. In the ribose degrada-
tion assay, the hydrogen atoms on the five carbon atoms
(C5’ ~ C1’), H-5’ ~ H-1’, respectively, of the 2’-deoxy-
D-ribose sugar ring are all essentially available for reac-
tion, i.e., hydrogen abstraction, with OH, a neutral, non-
discriminating and diffusible radical species, leading to
DNA degradation. However, in duplex DNA, such as the
plasmid DNA used in the DNA integrity assay, the pref-
erence for individual hydrogen atoms is seen to be in the
order: H-5’ > H-4’ > H-2’ H-3’ > H-1’, and this order
of reactivity correlates well with the solvent accessibility
of the individual hydrogen atoms in the sequence studied
[17,21,22]. This is verified by one interesting result from
the work of Close and coworkers, who irradiated single
crystals of 2’-deoxy-guanosine-5’-monophosphate at low
temperature and used ENDOR spectroscopy to detect
radicals at all five carbon atoms [23]; radicals were not
observed in irradiated duplex DNA [24]. Therefore, in
the DNA integrity assay, the number of hydrogen atoms
on each DNA sugar ring accessible to OH is indeed very
limited and OH is instead presumably scavenged by the
ingredients of the aqueous extracts of shallot powder
examined in this study.
The results of the EPR experiments show that lyophi-
lized shallot aqueous extract scavenges more OH than its
lyophilized garlic counterpart. The shallot extract demon-
strates a stronger radical-scavenging ability against ABT•+
than that of garlic, as previously noted. In terms of radi-
cal scavenging ability, polyphenols, not diallyl sulfide, in
allium vegetables are predominantly responsible for the
initial reduction of the ABT•+ [13]. The total polyphenol
content of dry bulbs of shallot and garlic are 674 ± 37.0
and 256 ± 2.60 mg/100 g [25], respectively, which sug-
gest that the difference in OH-scavenging ability be-
tween shallot and garlic aqueous extracts is most likely
due to the difference in their polyphenol content.
The results obtained in the EPR experiments are simi-
lar to those in the DNA integrity assay, i.e., lyophilized
shallot aqueous extracts exhibit scavenging activity against
OH. In the DNA integrity assay, as described above, the
lower solvent accessibility of the hydrogen atoms on the
sugar moieties of the duplex DNA is believed to be the
cause of OH scavenging by shallot powder aqueous ex-
tracts. Similarly, the EPR experiments show that the
OH-trapping efficiency of DMPO is only 35%, due pri-
marily to the competition between hydrogen abstraction
of the 1-pyrroline ring and OH addition to the C = N of
DMPO to generate EPR-active DMPO-OH [26]. This
means that OH is more likely to be scavenged by the
polyphenols in the aqueous extracts of shallot and garlic
powders. The low reactivity of DMPO toward OH can
also be explained by the fact that the nitrogen atom in the
1-pyrroline ring of DMPO is positively charged, making
hydrogen atom abstraction and C = N addition, both me-
diated by the electron-deficient OH, very difficult.
In summary, this study shows that shallot can be pre-
served in powder form for long-term storage, using three
different protocols. Additionally, the results show that
shallot powder aqueous extracts demonstrate either strong
reducing power or OH-scavenging ability, depending on
the assays conducted. Since shallot has been used as a
food additive for centuries, the results presented here
may increase the beneficial effects of shallot.
Copyright © 2012 SciRes. OJAppS
5. Acknowledgements
The authors would like to thank the Department of Bio-
technology at Ming Chuan University. They would also
like to thank Ms. Yuo-Chi Chen of the Department of
Chemistry at National Tsing Hua University (Hsinchu,
Taiwan) for her kind assistance in EPR data collection.
Finally, they are grateful to Mrs. Judy Wu and Dr. Mi-
chael McGarrgle, for proof-reading of the manuscript.
This study was supported by the Undergraduate Student
Research Fund of the Department of Biotechnology at
Ming Chuan University.
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