Antimutagenicity, Antioxidant Activities, And Total Phenolic Contents Of Five Mild
Thai Soups
Panukorn Boonsala1,*, Kaew Kungsadalumpai#, Kalyarat Kruawan, Pongtorn Sungpuag
1
Master of Science Program in Food and Nutritional Toxicology, Institute of Nutrition,
Mahidol University, Salaya, Phutthamonthon, Nakornpathom 73170, Thailand
*e-mail: p.boonsala@hotmail.com, #e-mail: kaew.kan@mahidol.ac.th
Abstract
Most mild Thai soups contain vegetables and spices; this makes them easily to be
modified for better health of the consumer. Five soups were prepared using the same soup
stock. Certain ingredients that made them different were tofu, white radish, green cabbage,
shiitake mushroom and pork blood jelly. The soup stock was cooked with each certain
ingredient listed above (150 g). Then, it was homogenized, lyophilized and determined for
antioxidant activities and total phenolic content. The free radical (DPPH) scavenging activity
ranged from 1.50 (pork blood jelly soup) to 3.27 (tofu soup) mM Trolox equivalent/g, the
ferric reducing antioxidant power (FRAP) ranged from 0.25 (pork blood jelly soup) to 2.81
(shiitake mushroom soup) µM ferrous/g, and total phenolic content ranged from 0.44 (pork
blood jelly soup) to 2.00 (shiitake mushroom soup) mg gallic acid equivalent/g. In the
somatic mutation and recombination test using Drosophila melanogaster, each sample was
mixed with Drosophila medium to obtain an experimental medium containing 12.5, 25 or 50
percents of sample in order to test its mutagenicity; the results indicated that none was
mutagenic. Bringing up the tester organism on each experimental medium containing 20 mM
urethane showed that only the soup consisted of tofu, white radish, green cabbage, or shiitake
mushroom reduced the mutagenicity of urethane. The percentage of inhibition was within the
range from 4.3% (pork blood jelly soup) to 33.7% (tofu soup). It was proposed that the
ingredients of soups might contain various antioxidants that modulated the metabolizing
enzymes resulting the reduction of wing spots induced by urethane. In conclusion, mild Thai
soups are good for health concerning consumer because they contain some antioxidants
associated with antimutagenicity.
Keywords: antimutagenicity, antioxidant activities, Drosophila melanogaster, mild Thai
soups, urethane
Introduction
Regular consumption of antimutagens in diet may be the most effective way of human
cancer prevention. Antimutagens occur in natural sources such as fruits and vegetables; they
may act as inhibitors of either the initiation or promotion of carcinogenesis [1]. It is well
known that Thai dishes which are a cultural inheritance transferred to and from generations
have various kinds of fruit and vegetable as the ingredients. The taste of each Thai dish is the
appropriate combination of sour, salty, sweet and spicy. Tom-Yum, an example of Thai
ethnic food, is consumed worldwide due to its tastes, colors and health effect. The major
ingredients of the soup are lemon grass, kaffir lime leaves, garlic, shallot, galangal root, and
chili [2]. However, some individuals dislike chili-based sensations at all concentrations [3];
then, they turn to love mild Thai soups. Therefore, we proposed to investigate the antioxidant
activities, total phenolic compounds, and the antimutagenicity against urethane in Drosophila
melanogaster of five mild Thai soups.
Methodology
Chemicals and Reagents
Urethane, 2, 4, 6-tripyridyl-s-triazine (TPTZ), ferric chloride hexahydrate, and ferrous
sulfate heptahydrate were purchased from Sigma Chemical (St. Louis, Missouri, USA). Fluka
Chemika (Buchs, Switzerland) supplied 2, 2- diphenyl-1-picrylhydrazl (DPPH), gallic acid
and Folin-Ciocalteu reagent. Trolox was purchased from Aldrich Chemical (Milwaukee, WI,
Germany). Other chemicals were of laboratory grade.
Sample Preparation
Pork bone (500 g), black pepper (3 g), salt (12 g), soybean sauce (180 ml) and fish
sauce (45 ml) and water (5,500 ml) were boiled for 45 minutes to be soup stock. A portion of
soup stock (840 ml) was mixed with minced pork (200 g) and a certain ingredient (150 g of
tofu, white radish, green cabbage, shiitake mushroom or pork blood jelly). Each mixture was
boiled for 12 min; then, it was added with coriander leaves (4 g) and chopped scallion (4 g).
A concurrent control soup containing everything but certain ingredient was prepared. Each
finished soup was finely homogenized, lyophilized and stored in a refrigerator until used.
Determination on Antioxidant Activities and Total Phenolic Content
Each dried sample (1 g) was extracted with 80% ethanol (10 ml) at 40ºC on an orbital
shaker set at 120 rpm for 2 h. The solution was filtered through Whatman filter paper No.1
and collected into a glass tube until used. The ethanolic extract was assayed for DPPH free
radical scavenging activity, ferric reducing antioxidant power (FRAP) and total phenolic
content as suggested by Kruawan and Kangsadalampai [4].
Somatic Mutation and Recombination Test
In the mutagenicity testing, each freeze-dried sample was mixed with the mixture of
dry components of standard Drosophila medium [5] to have the final 12.5, 25 or 50% w/w
sample in the medium; the mixture was added with water and proceeded to be the
experimental medium. The mutagenicity of each sample (in the experimental medium) was
assayed as described by Graf et al. [6]. Virgin females of Oregon wing flare strain
(ORR/ORR;flr3/In (3LR) TM3, ri pp sep l (3) 89Aa bx34e e Bds, Ser) were mated with males of
multiple wing hair strain (mwh/mwh) on standard medium to produce trans-heterozygous
larvae of improved high bioactivation cross; both strains were kindly provided by Professor
U. Graf (University of Zurich, Switzerland). One hundred larvae were transferred to each
experimental medium and maintained at 25+1oC until pupation. The standard medium was
used as a medium for the negative control group and the standard medium containing 20 mM
urethane was used as a medium for the positive control group. The surviving adult flies
bearing the marker trans-heterozygous (mwh+/+flr3) indicated with round wings were
collected. Wings were removed and mounted on a glass slide. The number of spots per wing
was scored under a compound microscope. Induction frequencies of wing spots of sample
treated groups were compared with that of the deionized water negative control group. A
multiple-decision procedure was used to decide whether a sample was positive, weak
positive, inconclusive or negative mutagenicity as described by Frei and Wurgler [7].
The highest concentration of sample in the experimental medium that provided the
surviving adult flies more than 50% was selected for the determination of its
antimutagenicity. In the determination, 20 mM urethane was substituted for deionized water
in the experimental medium; then the experiment was performed as did in the mutagenicity
determination. The antimutagenicity of each sample was determined from the percentage of
inhibition calculated as following:
Percentage of inhibition = ((A-B)/A) x 100
Where A is an average total spots per wing induce by urethane, B is an average total
spots per wing induce by urethane in the presence of each sample. It was proposed that
percentage of inhibition between 0–20%, 20–40%, 40–60% and higher than 60% were
classified as negligible, weak, moderate and strong antimutagenicity, respectively.
Results
Antioxidant Activities and Total Phenolic Content of Each Mild Thai soup
The antioxidant activities and total phenolic content of the ethanolic extracts from
each mild Thai soup are shown in Figure 1. It was revealed that most soups contained
antioxidants and phenolic compounds. The antioxidant activities and total phenolic contents
of the extract of pork blood jelly soup and white radish soup were lower than that of other
soups (control soup, green cabbage soup, shiitake mushroom soup, and tofu soup) in all
assays. Moreover, the antioxidant activities and total phenolic content of the extract of
shiitake mushroom soup had the highest antioxidant activity determined using FRAP assay
(2.81 µM Fe(II)/g dry weight of sample) and the highest total phenolic content (2.00 mg
gallic acid equivalent/g dry weight of sample)
The DPPH free radical scavenging activity of each sample ranged from 1.50 (pork
blood jelly soup) to 3.27 (tofu soup) mM Trolox equivalent/g dry weight of sample (Figure
1a). The FRAP values of each sample ranged from 0.25 (pork blood jelly soup) to 2.81
(shiitake mushroom soup) µM ferrous tripyridyltriazine/g dry weight of sample (Figure 1b).
The total phenolic content of each sample ranged from 0.44 (pork blood jelly soup) to 2.00
(shiitake mushroom soup) mg gallic acid equivalent/g dry weight of sample (Figure 1c).
Somatic Mutation and Recombination Test
In the mutagenicity evaluation of each mild Thai soup, the number of total spots per
wing of surviving adult flies derived from larvae brought up on Drosophila medium
containing each sample (50% w/w) was not significantly different from that of the flies
brought up on the negative control medium (data not shown). This indicated that all samples
contain none or undetectable mutagens. The percentages of surviving adult flies fed on each
experimental medium were higher than 50%; thus, it indicated that none of the samples was
too toxic for further antimutagenicity testing.
The antimutagenicity of each mild Thai soup is shown in Figure 2. The number of
total spots per wing of adult flies obtained from each experimental medium containing 20
mM urethane was compared with that of adult flies obtained from positive control group in
order to derive the percentage of inhibition on urethane mutagenicity. The result showed that
almost soups but pork blood jelly soup decreased the mutagenicity of urethane. The
percentage of inhibition on urethane mutagenicity of each sample was within the range of
4.3% (pork blood jelly soup) to 33.7% (tofu soup).
Discussion and Conclusion
The result of the present study confirmed the safeness of five mild Thai soups to
consumers and also revealed that they were good sources of antioxidants including phenolic
compounds. Therefore, the protecting effect against urethane of four soups, namely tofu soup,
shiitake mushroom soup, green cabbage soup, and white radish soup may relate to the
presence of antioxidants and phenolic compounds in the main vegetable of the soup. This
might give the clue on the mechanism of antimutagenicity against urethane of each soup. It is
known that urethane is metabolically activated by cytochrome P-450 enzyme system [8] to be
vinyl epoxide, the carcinogenic active metabolite [9] that is further detoxified with
glutathione-S-transferase (GST) conjugation [10]. Some antioxidants of the samples might
inhibit cytochrome P450 system or induce glutathione-S-transferase in the same way as
instant coffee, a good source of antioxidants [11], co-administered with urethane to
Drosophila larvae [12] inhibited the activities of cytochrome P450 enzymes leading to a
reduction in the rate of bioactivation of urethane. It was proposed that the antimutagenicity of
tofu soup might be due to the induction of glutathione-S-transferase activity by genistein.
Figure 1.The antioxidant activities and total phenolic content of ethanolic extracts from each Thai soup.
(a) DPPH assay, (b) FRAP assay, and (c) total phenolic content
Figure 2.The inhibitory effect of each mild Thai soup against urethane (20mM) induced somatic mutation
and recombination in Drosophila melanogaster derived from trans-heterozygous (mwh+/+flr3) larvae.
This phytochemical is a biologically active component in soy that can modulate gene
expression and the activity of enzymes involving in antioxidant defence and the metabolism
of xenobiotics including nicotinamide adenine dinucleotide phosphate (NADPH), quinone
oxidoreductase 1, and glutathione-S-transferase in rat liver [13]. The polysaccharide of
shiitake mushroom was suspected for the moderate increase of glutathione-S-transferase
activity in in vitro model [14]; therefore, this compound in shiitake mushroom soup should
induce glutathione-S-transferase activity that detoxified the active species of urethane. The
main vegetable in green cabbage soup is a member of the family Brassicaceae which
generally contains isothiocyanates and indoles; these two phytochemicals were shown to
attenuate the effects of polycyclic aromatic hydrocarbons and nitrosamines via induction of
glutathione-S-transferase and inhibition of cytochrome-P450 in rodents [15]. In addition,
white radish soup also contains the main vegetable that belongs to the family Brassicaceae;
thus, the modifying of detoxifying enzymes of the testing organism was possible. Hanlon et
al [16] reported that the 70% acetone solution extracts of Spanish black radish significantly
induced the phase I cytochrome P450 system (CYP1A1 and CYP1B1) and the phase II
detoxification enzymes such as quinone reductase, heme oxygenase, and thioredoxin
reductase in the human hepatoma cell line. It is, therefore, a need to determine whether white
radish has the same enzyme induction activity as that of Spanish black radish.
In addition, during the metabolism of urethane it was documented that Nhydroxyurethane, a urethane metabolite [17, 18] occurred and was hydrolyzed by esterase to
generate hydroxylamine which exerted its carcinogenic effect in multiple organs via
generating O2·- and NO· to cause oxidation and depurination of DNA [19]. Therefore, the
antioxidant activities of tofu soup, shiitake mushroom soup, green cabbage soup, and white
radish soup might scavenge O2·- and NO·; this should be the benefit of antioxidants in mild
Thai soups.
The present investigation showed that pork blood jelly soup and control soup were
classified as negligible in diminishing the mutagenicity of urethane since their inhibitory
effects were less than 20% of the positive control. The activity as such might be due to the
presence of small amount of some antimutagens in spices such as coriander [20] in each mild
Thai soup. In addition, the original idea of this investigation was due to the fact that hemin of
pork blood jelly should directly trap urethane as it was able to trap heterocyclic amines and
prevented their mutagenicity [21]. Therefore, the failure of pork blood jelly in inhibiting
urethane mutagenicity should come from the difference of chemical structure of mutagenic
species. Further studies using different testing methods with different positive mutagens
might elucidate the antimutagenicity of pork blood jelly soup.
In conclusion, mild Thai soups (control soup, green cabbage soup, white radish soup,
shiitake mushroom soup, tofu soup, and pork blood jelly soup) are safe for most consumers.
They are good for health due to the fact that they contain some antioxidants associated with
antimutagenicity against urethane in somatic mutation and recombination test. Further studies
should be used other testing methods with other positive mutagens for pork blood jelly soup
that might express its antimutagenicity against other mutagens.
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