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Food and Chemical Toxicology 42 (2004) 659–666

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Antimutagenic, antioxidant and free radical scavenging activity of


ethyl acetate extracts from white, yellow and red onions
Mi-Yae Shona, Sang-Do Choib, Goon-Gjung Kahngb, Sang-Hae Namb, Nak-Ju Sunga,*
a
Department of Food and Nutrition, Institute of Agriculture & Life Science, Gyeongsang National University, 900 Gaza-dong chinju city,
South Korea
b
Department of Food Processing, Jinju National University, 660-758, Chilam-dong chin-ju city, South Korea

Received 31 May 2003; received in revised form 15 November 2003; accepted 8 December 2003

Abstract
The beneficial effects of red, yellow and white onion extracts have been assessed by antioxidant activity and antimutagenic
activity. And the effects compared to BHT and ascorbic acid. Total phenolic compounds and flavonoids in onion extracts were
determined. Yellow onion extract had more organic acid and free sugar than those detected in the white and red onion extract. The
scavenging activity of DPPH radical and H2O2 were increased depending on the concentration. The antioxidant activities using b-
carotene-linoleate system and reducing power were increased but the effect was small to that of BHT and ascorbic acid. After
digested, extracts showed antimutagenic activities, and it seems that they inhibit the mutagenicity for digesting. This study
demonstrated that the antimutagenicities and antioxidant properties of ethyl acetate extract against mutagens were related to their
phenols and flavonoids, which are heat stable and losses digestive juices are relatively low.
# 2003 Elsevier Ltd. All rights reserved.
Keywords: Antioxidant activity; Antimutagenic activity; Phenols; Flavonoids; Onion extracts; Digestive solutions

1. Introduction This protective effect is attributed to flavonoids and


phenolic compounds present in these foods. To investi-
Flavonoids and phenolic compounds are occurring in gate the mechanisms of these effects in animal bodies,
food plants and are a common component in the human several studies have been conducted to determine their
diet. Food-derived flavonoids and phenolic compounds absorption and distribution. Onions have been shown
such as the flavonols quercetin, kaempferol, gallic acid to contain large amounts of flavonoids, and constitute
and myricetin have been reported to exhibit a wide one of the major sources of flavonoids in diets (Knekt et
range of biological effects, including antibacterial, anti- al., 1996). Onion, a vegetable member of the genus
viral, anti-inflammatory, antiallergic actions (Huang et Allium, has been reported to promote cardiovascular
al., 1983). In addition, flavonoids inhibit lipid peroxi- health exhibiting decreased rates of atherosclerosis or
dation and exert these effects as antioxidants, free radi- thrombotic disease in populations with increased onion
cal scavengers, and chelators of divalent cations. intake (Kendler, 1987). These beneficial effects of onion
Epidemiological research on the relation between flavo- have been attributed to its ability to inhibiot platelet
noid intake and disease risk in humans is needed to aggregation and thromboxane formation (Srivastava,
support the findings of these experimental studies. Epi- 1986). However there is little chemopreventive inform-
demiological studies investigating relations between diet ation on effects of extracts of three varieties of onions.
and disease, a protective effect of the consumption of This research is needed to investigate the character the
vegetables and fruits on various forms has been studied components in onions.
(Steinmetz and Potter, 1991). Flavonoids are low molecular weight polyphenolic
substances based on the flavan nucleus. The biochemical
* Corresponding author. Tel.: +82-55-751-5975; fax: +82-55-751-
activities of flavonoids and their metabolites depend on
5974. their chemical structure and the relative orientation of
E-mail address: nuruksmy@hanmail.net (N.-J. Sung). various moieties on the molecule. They are hydrolyzed
0278-6915/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2003.12.002
660 M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666

by intestinal flora to produce the biologically active were purchased from Sigma Chemical Co. (St. Louis,
aglycone (sugar-free flavonoid). There is now increasing Mo, USA). IQ was purchased from Wako Chemical
evidence about absorption of flavonoids and some of Inc. The Salmonella typhimurium strains TA98 and
them have been detected in human plasma and other TA100 were purchased from KCTC (Korean Collection
biological fluids (Bourne and Rice-Evans, 1999; Pietta for Type Cultures). Aroclor 1254-induced hepatic S9
& Simonetti, 1999; Terao, 1999). In the human study, was made for the activation system in the case of IQ.
the absorption of orally administered quercetin was Agar and Nutrient Broth No.2 were purchased from the
24%, while the absorption of quercetin glycosides from Difco Laboratories (Detroit, USA) and Oxoid (Hamp-
onions was 52% (Hollman et al., 1995). Dietary inhibi- shire, UK), respectively.
tors of mutagenesis and carcinogenesis are of particular
interest because they may be useful for human cancer 2.2. Preparation of onion extracts
prevention on recently, several flavonoids and phenolic
compounds have been demonstrated to have an anti- The onions varieties white skinned (Albion), yellow
mutagenic effect on various mutagens or carcinogens skinned (Rijnsburger) and red skinned (varity Red
(Francis et al., 1989; Chang-qi et al., 1994; Birt et al., Baron) were purchased from local markets. Each onion
1986). Also dietary flavonoids, a group of polyphenols, was skinned, chopped and lyophilized. The lyophilized
suppressed the metabolism of the widespread food car- onions were ground to a fine power. Ground onion
cinogen 2-amino-3-methylimidazo-[4,5-f]quinoline(IQ) powers (50 g) were extracted with 85% ethyl acetate
(Edenharder et al., 1997), in the presence of a hepatic room temperature for 12 h (3 times with 500 ml),
activation system derived from Aroclor 1254-treated respectively. Ethyl acetate extract contain hydrophilic
rats, is a typical heterocyclic amine mutagen found in and lipophilic nature. It is of general interest to measure
cooked foods. The ellagic acid scavenged the reactive the total antioxidant activity. The extracts were con-
intermediates of polycyclic aromatic hydrocarbons centrated in a vacuum evaporator (EYELA, Japan) at
(Sayer et al., 1982). Polyphenols can also impair the below 40  C. All of the concentration were lyophilized
promotion stage of carcinogenesis as a result of their and stored at 20  C.
antioxidant activity (Vinson et al., 1995).
Plant flavonoids are known to be easily degraded in 2.3. Determination of phenolic compounds
alkaline solutions (Tamura and Yoshino, 1997). Human
intestinal and pancreatic juices also are known to be The contents of total phenols were analyzed by the
mildly alkaline, with pH values of 8.3 and 7.0 to 8.5, method of Folin and Denis (Folin and Denis, 1915),
respectively. Therefore, in this study, we determined the reading samples on a UV/Vis spectrophotometer at 760
antimutagenic activity and antioxidant activity with nm. The each extract was mixed with 5 ml of Folin–
onion extracts. Furthermore, we also investigated the Denis phenol reagent, 10 ml of Na2CO3 and diluted by
antimutagenic activity of onion extracts after digestion a factor 100 with distilled water. The total phenol con-
in simulated gastric and intestinal juice under various tent of each solvent extract was estimated by compar-
conditions with mutagen. The aim of the present study ison with a standard curve generated from analysis of
is a need to know the absorption of flavonoids and caffeic acid.
phenolic acids whether they are loss or not in the simu- The content of total flavonoids was analyzed color-
lated gastric and intestinal juices. If they will absorb and imetrically by the Davis method using myricetin as a
metabolize, they have been shown to inhibit about standard (AOAC, 1995). The each extract (0.01 g) was
mutagen. mixed 30 ml of distilled water and 30 ml of methanol.
After boiling for 30 min in 90  C, cooled and adjusted
100 ml of distilled water. Diluted solution was filtered. 1
2. Materials and methods ml of filtrared solution was mixed with 10 ml of diethy-
lene glycol and 1 ml of 1N-NaOH. The absorbances of
2.1. Materials sample and blank were measured at 420 nm after stand
for 30 min in the dark at room temperature.
All solvents used were of HPLC grade, Reagents and Organic acid was determined with HPLC analysis
chemicals were purchased from Sigma-Aldrich, Wako (AOAC, 1995). The each extract (1 g) was dissolved in
and Fluka. b-carotene, linoleic acid, polyoxyethylene distilled water (50 ml) and filtered through 0.45 mm fil-
sorbitan monopalmitate (Tween 40), buthylated hydro- ter. The filtrate was stirred for 30 min. The filtrate (0.2
xytoluene (BHT), l-ascorbic acid, pepsin(from hog sto- ml) was injected into the HPLC apparatus.
mach), pancreatin(from porcine pancreas), 2,2- Chromatography was performed on a m-Bondapack
diphenyl-picrylhydrazyl(DPPH), liloeic acid, EDTA, C-18 column (Water-Millipore Corporation. Milford,
ferric chloride, hydrogenperoxide (H2O2, 30%, v/v), MA, USA). The mobile phase was water/methanol
vanillin, potassium ferricyanide, DMSO and MNNG 10:80 containing 2% of acetic acid. The flow-rate was
M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666 661

set at 1.0 ml/min. Detection was effected at 330 nm. The prepared for back ground subtraction. Antioxidant
peak assignments were based on the retention times of activity (AA) was calculated using the following
the single compound. Calibration graphs were plotted equation:
showing a linear relationship between concentrations
versus peak-areas for reference compound. AA ¼ b-carotene content after 2 h of assay=

Free sugar was determined with HPLC analysis initial b-carotene content  100
(AOAC, 1995). The extract (1 g) was dissolved in dis-
tilled water (30 ml) and centrifuged at 8000 rpm for 20
min. The supernatant make up 50 ml with distilled 2.6. Reducing power activity
water and filtered through 0.2 mm filter. The filtrate was
passed with sep-pak C18 and injected into the HPLC The reducing power of extracts was determined by the
apparatus (Water-Millipore Corporation. Milford, MA, method of Yen and Duh (1993). Different concentra-
USA). Chromatography was performed carbohydrate tions of extracts were mixed with 2.5 ml of phosphate
column. The mobile phase was 80% acetonitrile. The buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium
flow-rate was set at 2.0 ml/min. The peak assignments ferricyanide. The mixtures were incubated for 20 min at
were based on the retention times of the single 50  C. After incubation, 2.5 ml of 10% trichloroacetic
compound. acid were added to the mixtures, followed by cen-
trifugation at 650g for 10 min. The upper layer (5 ml)
2.4. DPPH radicals scavenging activity was mixed with 5 ml of distilled water and 1 ml of 0.1%
ferric chloride and the absorbance of the resultant solu-
The scavenging activity of onion extracts by DPPH tion was measured at 700 nm.
radicals was measure according to the method of Shimada
et al. (1992). Ethanolic solutions of DPPH (104) and 2.7. Hydrogen peroxide scavenging activity
onion extracts solutions were mixed so that the final
mass ratios were extracts: DPPH.=5.5:1 and reference The extract was dissolved in 3, 4 ml of 0.1 M phos-
compounds: DPPH.=0.5:1. The samples were incu- phate buffer(pH 7.4) and mixed with 600 ml of 43 mM
bated for 15 min in the dark was at 30  C and the solution of hydrogen peroxide. The absorbance value
decrease in absorbance at 517 nm was measured against (at 230 nm) of the reaction mixture was recorded from 0
ethanol using a spectrophotometer. Ethanol was used to min to 40 min and then at every 10 min. For each con-
zero the spectrophotometer; a blank sample containging centration, a separate blank sample was used for back
the same amount of ethanol and DPPH. was prepared ground subtraction (Ruch et al., 1989).
and measured daily, stored in a flask covered and kept
in the dark at 4  C between the measurements. All 2.8. Preparation of the gastric and intestinal juice
determinations were performed in triplicate.
The simulated gastric and intestinal juice was pre-
2.5. Antioxidant assay using -carotene linoleate model pared as follows (USP 23).
system The gastric fluid consisted of 2.0 g of NaCl and 3.2 g
of Pepsin, dissolved in 7.0 ml of HCl, And water was
The antioxidant activity of extracts was evaluated by added to make 1000 ml. The pH of the gastric fluid was
the b-carotene linoleate model system (Miller, 1971). A pH 1.2. The intestinal fluid consisted of 6.8 g of
solution of b-carotene was prepared by dissolving 2 mg KH2PO4, dissolved in 250 ml of water, 190 ml of 0.2N
of b-carotene in 10 ml of chloroform. Two mililitres of NaOH, 400 ml of water and 10.0 g of pancreatin. The
this solution were pipetted into a 100 ml round bottom pH of the intestinal fluid was pH 7.5  0.1.
flask. After chloroform was removed under vacuum, 40
mg of purified linoleic acid, 400mg of Tween 40 emulsi- 2.9. Preparation of digested sample solution
fier, and 100 ml of aerated distilled water were added to
the flask with vigorous shaking. Aliquots (4.8 ml) of this The lyophilized onion extracts were each submitted
emulsion were transferred into a different test tubes to gastric or intestinal juice and a mutagen such as
containing different concentration of the extract. BHA IQ or MNNG. A 0.1 ml aliquot of each solution of
was used for comparative purposes. As soon as the extract and mutagen was subjected to an equal volume
emulsion was added to each tube, the zero time absor- of gastric juice for 30 min. Another 0.1 ml aliquot of
bance was measured at 470 nm using a spectro- each solution of extract and mutagen submitted to
photometer (CE2021, CECIL, England). The tubes an equal volume of intestinal juice for 2 h. This pro-
were placed at 50  C in a water bath. Measurement of cess was performed in triplicate. Digested samples
absorbance was continued until the colour of b-carotene were stored at 20  C until analysis in the mutagenic
disappeared for the blank, devoid of b-carotene, were activity.
662 M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666

The antimutagenic activity of the lyophilized onion In the DPPH test, the onion extracts were able to
extracts used in the experiments with the in vitro model reduce the stable radical DPPH to the yellow coloured
was tested by Ames test (Maron and Ames, 1983). The diphenylpicrylhydrazine. Methanol and hot water
antimutagenicities were used in the concentration of 3 extracts showed radical scavenging activity with 85% of
mg per plate. As a mutagenic agent for TA98 and 500 mg. The scavenging activity of radical on onion
TA100 was 0.2 mg for IQ and 0.1 mg for MNNG. For extracts and BHT is presented in Fig. 1.
each sample three agar plates were used and the number Hydrogen peroxide scavenging activity of the extract
of revertants per plate was counted with a colony is presented in Fig. 2. Scavenging activity of red onion
counter. Results of the Ames test were expressed as extract (10 mg/ml) and BHT(10 mg/l), l-ascorbic acid(10
mean number of revertants per triplicate plates, cor- mg/ml), reference compound, were 81% and 90%, 80%
rected for spontaneous revertants. of the initial concentration, respectively.
Fig. 3 shows the reducing power of the extracts using
2.10. Antimutagenic activity of digested onion extracts the potassium ferricyanide reduction method. At 1 mg/
ml concentration, the extract obtained using red onion,
The antimutagenic activity of the digested onion yellow onion and white onion showed absorbances of
extracts used in the experiments with the in vitro model 0.17, 0.12, and 0.12, respectively. The l-ascorbic acid,
was tested by Ames test (Maron and Ames, 1983). This reference compound, exhibited the high reducing activ-
procedure is same that used to measure antimutagenic ity of 2.94 in 300 mg/ml.
activity of the lyophilized onion extracts. All experi- Fig. 4 shows the antioxidant activity of the extracts as
ments were performed in triplicate. Data presented in measured by the bleaching of b-carotene. Extracts in red
the tales are means from three independent series. onion, yellow onion and white onion as well as BHT,

3. Results

In total phenols and total flavonoids contents of


Table 1, red onion gave a high yields. The contents of
organic acids and free sugars are shown in Table 2. In
total free sugars content, all onion extracts gave about
600 mg/1 mg. In total organic acids content, yellow
onion gave a high yields.

Table 1
Contents of total phenolic compounds and flavonoids in ethyl acetate
extracts from three kinds of onion

(Dry base, m/mg)a


b
Onions Total phenols Total flavonoidsc
Fig. 1. Scavenging activities of ethyl acetate exrtracts of onion against
White 115 4 0.412 1,1-diphenyl-2-picrylhydrazl radical.
Red 133 7 0.511
Yellow 107 15 0.22
a
Each sample analyzed in triplicate
b
Total phenol contents based a standard curve generated by caffeic
acid.
c
Total flavonoids contents based a standard curve generated by
myricetin.

Table 2
Contents of total organic acids and free sugars in ethyl acetate extracts
from three kinds of onion

(Dry base, m/mg)


Onions Total organic acids Total free sugars

White 190.95a 629.14


Red 164.514 626.1212
Yellow 268.711 542.510
Fig. 2. Scavenging activities of ethyl acetate exrtacts of onion on
a
Data are presented as the meanS.D. of triplicate determinations. hydrogenperoxide.
M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666 663

l-ascorbic acid itself, were found to give the antioxidant al., 2001). Amounts of 3 mg per plate of onion extracts
activity of 27%, 20%, 22%, 89%, and 37% at 1 mg/ml were sufficient to inhibit the mutagenicity induced by
concentration, respectively. any of the IQ and MNNG concentrations used. These
The onion extracts showed antimutagenicity against results suggest that ethyl acetate extracts have anti-
IQ and MNNG in Salmonella typhimurium TA98 and mutagenic effects on both IQ and MNNG.
TA100 (Table 3). Welsh onion has already demon- In Tables 4 and 5, digested onion extracts also showed
strated its effective antimutagenic activity (Huifeng et antimutagenicity. In all cases, concentration dependent
inhibition was observed. There were no significant dif-
ferences in antimutagenicity on the digested onion
extracts.

4. Discussion

Flavonoids and phenolic acids are the most pre-


dominant components of onion. These compounds have
antimutagenic potential activity. Onion extracts also
showed radical scavengers and antioxidant activities.
The onion extracts had phenolic hudroxyl groups in the
structure and antioxidants of phenolic groups have been
recognized to function as electron or hydrogen donors
(Shahidi and Wanasundara, 1992). The role of anti-
oxidants has attracted much interest with respect to
their protective effect against free radical damage that
Fig. 3. Reducing power of ethyl acetate extracts of onion. may be the cause of many diseases including cancer
(Nakama et al., 1993). The antioxidative effect of onion
extract is mainly due to the phenolic components, such
as the flavonoids (Pietta et al., 1998). Some flavonoid
and non-flavonoid compounds have been reported to
also show alkylperoxyl radical scavenging activity thus
reducing radical-mediated pathogenesis, e.g. carcino-
genesis (Sawa et al., 1999).
Thus, the DPPH radical scavenging activity of onion
extracts may be mostly related to their phenolic
hydroxyl group. Also the concentration of hydrogen
peroxide in water may occur according to the phenolic
compounds and flavonoids. Since phenolic compounds
present in the extract are good electron donors, they may
accelerate the conversion of H2O2 to H2O (Ruch et al,
1984). As the good electron donors, they show the redu-
Fig. 4. Antioxidant activities of ethyl acetate extracts of onion using cing power. In Fig. 4, the antioxidant activity of car-
b-carotene linoleate model system. otenoids is based on the radical adducts of carotenoid

Table 3
Antimutagenic activity of ethyl acetate extracts from three kinds of on ion against IQ (0.02 mg, 0.2 mg/plate) and MNNG (0.1 mg, 0.01 mg/plate) on
Salmonella typhimurim TA98 and TA 100

Onions IQ MNNG
(3000 mg/plate)
Revertants/platea Percent inhibition (%) Revertants/plate Percent inhibition (%)

TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100

Control 152.712b 288.06 0 0 153.3 10 228.29 0 0


Red 59.35 154.52 61.12.9 46.43.3 57.0 13 127.15 62.8 2.5 44.3 3.6
White 47.37 145.010 69.00.8 49.75.6 57.2 6 133.02 62.7 2.3 41.7 0.4
Yellow 45.012 150.29 70.50.5 47.91.0 55.2 9 121.220 64.0 1.7 46.9 1.1
a
Triplicate plates were tested per dose per experiment.
b
Datas are presented as the mean S.D. of triplicate determinations.
664 M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666

with free radicals from linoleic acid. The linoleic acid Onions contain organosulfur compounds. Onion eth-
free radical attacks the highly unsaturated b-carotene anol extracts also contain lipophilic antioxidant prop-
models. The presence of carotenoids shows, not only a erties. It is known that onion oil contains dialkyl
decrease of the free radical concentration, but the disulfides and their oxides and thiols, which can trap
reduction of Fe3+ to Fe2+ by carotenoids. The pre- electrons from other systems. Thus it scavenges many
sence of different antioxidants can hinder the extent of free radicals including hydroxyl radicals (Klanns-Dieter,
b-carotene-bleaching by neutralizing the linoleate-free 1983). Organosulfur compounds are also known to be
radical and other free radicals formed in the system potent inducers of glutathione S-transferase (Hu et al.,
(Jayaprakasha et al., 2001). 1997). Onion extracts maintained their antimutagenic
All types of onions in our study contained antimuta- potential in vitro simulated digestion model. Thus,
gens and the extracts showed similar levels of anti- antimutagenic compounds from onion extracts were not
mutagenic activity. And antimutagenic effects of onion inactivated by gastric acid and intestinal juices and were
extracts depended on the mutagen and dose levels. readily available for absorption.

Table 4
Antimutagenic activity of ethyl acetate extracts from three kinds of onion against IQ (0.02 mg, 0.2 mg/plate) on Salmonella typhimurim TA98 and TA
100 under simulated gastric juice or intestinal juice

Onions Antimutagen Gastric juice Intestinal juice


(mg/plate)
Revertants/platea Percent inhibition (%) Revertants/plate Percent inhibition (%)

TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100


b
Red 0 228.35 307.33 0 0 217.07 602.31 0 0
1000 95.72 272.06 58.3 7 11.5 1.0 180.04 232.615 17.1 0.9 61.4 1.0
2000 59.713 223.017 74.1 1.6 27.4 0.7 101.312 207.63 53.3 1.7 65.5 1.3
3000 30.310 128.611 86.8 2.6 58.2 0.7 41.39 155.69 81.0 2.0 74.2 0.8
White 0 178.33 161.05 0 0 217.08 264.05 0 0
1000 100.07 130.02 43.9 1.0 20.0 0.4 115.55 117.04 46.8 2.0 55.7 0.9
2000 78.019 78.7 12 56.3 0.4 50.9 1.1 67.716 114.013 68.8 0.7 56.8 0.8
3000 32.32 75.0 4 81.9 0.4 53.4 1.7 57.320 95.3 7 73.6 0.8 63.9 1.6
Yellow 0 593.35 489.05 0 0 351.56 308.03 0 0
1000 128.77 246.13 78.3 1,5 46.7 1.8 73.24 124.07 79.2 1.1 59.7 1.8
2000 94.719 176.011 84.0 1.5 64.0 0.8 69.72 105.225 77.9 0.9 65.8 1.3
3000 80.020 160.216 86.5 0.9 67.3 1.6 56.211 92.1 11 84.0 1.6 70.0 0.8
a
Triplicate plates were tested per dose per experiment.
b
Datas are presented as the mean S.D. of triplicate determinations.

Table 5
Antimutagenic activity of ethyl acetate extracts from three kinds of onion against MNNG (0.1 mg, 0.01 mg/plate) on Salmonella typhimurim TA98
and TA 100 under simulated gastric juice or intestinal juice

Onions Antimutagen Gastric juice Intestinal juice


(mg/plate)
Revertants/platea Percent inhibition (%) Revertants/plate Percent inhibition (%)

TA98 TA100 TA98 TA100 TA98 TA100 TA98 TA100


b
Red 0 226.23 258.52 0 0 476.61 276.012 0 0
1000 167.75 144.36 25.9 1.2 44.2 1.0 321.53 137.53 32.5 0.4 50.2 1.0
2000 140.011 124.015 38.1 1.6 52.0 0.4 255.723 124.55 46.3 0.7 54.9 0.9
3000 124.58 108.53 45.0 0.4 58.0 0.3 179.510 103.16 62.3 1.3 62.6 0.7
White 0 217.33 260.39 0 0 229.03 241.52 0 0
1000 133.12 200.35 38.7 1.0 23.1 0.7 144.37 189.37 37.0 1.1 21.6 2.1
2000 115.010 180.214 47.0 0.4 30.8 0.4 118.59 134.615 48.3 0.8 44.3 0.9
3000 109.217 105.220 49.7 1.3 59.6 1.4 100.310 102.06 56.2 0.3 57.8 0.2
Yellow 0 148.09 197.011 0 0 155.53 252.09 0 0
1000 124.04 163.67 16.2 0.6 17.0 1.7 129.321 194.023 16.8 0.9 23.0 0.6
2000 106.71 133.04 27.9 2.5 32.5 1.1 118.76 171.14 23.7 1.4 32.1 0.8
3000 48.017 101.77 67.6 0.2 48.4 0.3 84.79 119.32 45.5 1.5 52.6 1.6
a
Triplicate plates were tested per dose per experiment.
b
Datas are presented as the mean S.D. of triplicate determinations.
M.-Y. Shon et al. / Food and Chemical Toxicology 42 (2004) 659–666 665

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