Antioxidant Activities, Total Phenolic Content and Antimutagenicity of Ten Thai Chili
Pastes Prepared With Different Sour Fruit Juices
Kantika Prayoonruk1,*, Kaew Kangsadalampai#, Kalyarat Kruawan, Pongtorn Sungpuag
1
Master of Science Program in Food and Nutritional Toxicology, Institute of Nutrition,
Mahidol University, Thailand
*e-mail: kantika.pra@student.mahidol.ac.th, #e-mail: kaew.kan@mahidol.ac.th
Abstract
Thai chili paste is a condiment that adds spice and flavor to a wide range of dishes.
We prepared ten types of pastes. A standard condiment mixture (consisted of chili pepper,
garlic, shrimp paste, coconut sugar, pea eggplant and fish sauce) and one of the ten selected
sour fruit juices ( lime, kaffir lime, kumquat, lemon, mango, tamarind, bilimbi, mombin,
Madan and star gooseberry) were mixed and homogenized. A concurrent control without sour
juice was prepared to be the sample base (the eleventh paste). Analyzing for antioxidant
activities and total phenolic contents showed that the paste containing mombin had 3 .1 5 µM
TEAC/g in DPPH scavenging, 13.2 µM/g in FRAP assays and 1.18 mg GAE/g in total
phenolic content, respectively; the results were the highest values of all samples. In the
mutagenicity evaluation, each sample was added into the Drosophila medium in order to
have the final 6.25 % of sample in the experimental medium. All samples were found
negative in the somatic mutation and recombination test using mwh+/+flr3 Drosophila
melanogaster. The same test was also used to evaluate the antimutagenicity of each sample
against 20 mM urethane. The result demonstrated that none of the pastes reduced the
mutagenicity of urethane at the significant level. On the other hand, the paste containing lime,
Madan or lemon as well as the sample base significantly potentiated the mutagenicity of
urethane. The enhancing effect of each sample might be due to some natural compounds
could induce the catalytic activities of cytochrome P-450 enzyme system (phase 1) or
inhibited glutathione-S-transferase as well as decreased the amounts of glutathione of phase 2
detoxifying system in Drosophila melanogaster. Therefore, high consumption of Thai chili
paste should be done with caution because the data of the present investigation suggested that
it enhanced the mutagenicity of 20 mM urethane.
Keywords: Thai chili pastes, sour fruit juices, mutagenicity, antioxidant, SMART
Introduction
Thai chili paste is a condiment that adds spice and flavor to a wide range of dishes. It
can be used as a wonderful dip for all kinds of food such as fried Indo-Pacific Mackerel,
steamed or blanched vegetables etc. The main components are hot chili pepper, garlic,
coconut sugar and sour juice; such components are sources of antioxidants and phenolic
compounds [1, 2]. Generally, one of locally grown fruits such as Citrus fruit or Spondias fruit
that give sour taste is selected to give a specific flavor of each Thai chili paste. Sa-nga [3]
indicated that 98 % of Thai consumers favored to have chili paste as the member of their
meal. The main benefits to consumers of chili pastes mostly belong to herbs that have
nutrients and phytochemicals; however, no study has been conducted on the health protective
effect for consumers. Therefore, the aim of the proposed study was to investigate the effect of
ten types of chili paste on the antioxidant activity and antimutagenicity against urethane in
Drosophila melanogaster.
Methodology
Chemicals and Reagents
Urethane was purchased from Sigma Chemical (St. Louis, Missouri, USA). 2, 4, 6tripyridyl-s-triazine (TPTZ), Ferric chloride hexahydrate, and Ferrous sulfate heptahydrate
were purchased from Sigma Chemical (St. Louis, Missouri, USA). 2, 2- diphenyl-1-
picrylhydrazl (DPPH), Gallic acid and Folin-Ciocalteu reagent were purchased from
FlukaChemika (Buchs, Switzerland). Trolox was purchased from Aldrich Chemical
(Milwaukee, WI, Germany). Other chemicals were of laboratory grade.
Sample Preparation
Each chili paste contained 5 g chili pepper, 20 g garlic, 15 g shrimp paste, 30 g
coconut sugar, 20 g pea eggplant, 15 g fish sauce and 30 g sour fruit juice (lime, kaffir lime,
kumquat, lemon, mango, tamarind, bilimbi, mombin, Madan or star gooseberry).The
ingredients were mixed well in an electrical blender and kept refrigerated before it was
revealed for antioxidant activities, total phenolic content and antimutagenicity against
urethane.
Antioxidant Activity And Total Phenolic Content
Each sample (1g) was extracted with 80% ethanol (10 ml) at 37ºC for 30 minutes. The
solution was filtered through Whatman filter paper No. 1 and collected into a glass bottle.
Each ethanolic extract was assayed for DPPH free radical scavenging activity, ferric reducing
antioxidant power or FRAP and the total phenolic content as suggested by Kruawan and
Kangsadalampai [4].
Somatic Mutation and Recombination Test (SMART)
Freshly prepared standard Drosophila medium [5] was mixed with each sample to
have the final 6.25, 12.5, 25 or 50% (w/w) of the sample in the experimental medium. The
mutagenicity testing was performed as described by Graf et al [6]. Virgin females of Oregon
wing flare strain (ORR/ORR; flr3/TM3, 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 surviving adult flies bearing the marker transheterozygous (mwh+/+flr3) indicated with round wings were collected. Wings were removed
and mounted on a glass slide. 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 toxicity of each sample was determined from the data of
survival rate of adult flies. The wing spots data were evaluated using the statistical and a
multiple decision procedures described by Frei and Wurgler [7]. The result suggested that the
final 6.25% of sample in the experimental medium was suitable for further antimutagenicity
study; therefore, the experimental medium containing final 20 mM urethane was prepared in
order to evaluate the antimutagenicity of each sample and the experiment was proceed as
described above. The percentage of inhibition calculated as the following equation:
Percentage of inhibition = ((a-b)/a) 100
When a is the frequency of spots induced by urethane alone and b is the frequency of
spots induced by urethane in the presence of sample. It is proposed that percentages of
inhibition between 0-20 represent a negligible effect while expression of percent inhibitions
between 20-40, 40-60 and more than 60 are the evidences of weak, moderate and strong
antimutagenicity, respectively as suggested by Abraham [8].
Results
Figure 1 shows the antioxidant activities and total phenolic contents of all samples.
Lime, kaffir lime, kumquat, lemon, mango, tamarind, bilimbi, mombin, Madan or star
gooseberry, as a source of sour taste, could increase the free radical (DPPH) scavenging
activity of chili paste expressed as µM Trolox equivalent antioxidant capacity/g to be higher
than that of the sample base; the result is shown in Figure 1A. Free radical scavenging
activity of each sample is between 1.57 and 3.15 µM TEAC/g. In addition, all but mango
could increase the antioxidant expressed as FRAP values of the chili pastes to be higher than
that of the sample base; the values are between 3.0 and 13.2 µM/g (Figure 1B). Only mombin
Figure 1. Antioxidant activity in DPPH assay (A), antioxidant activity in FRAP assay (B) and
total phenolic content (C) of ethanol extract from each Thai chili pastes
.
juice could increase the content of phenolic compound to be 1.18 mg gallic acid/g (Figure
1C) which was higher than that of the sample base.
It was noted that only the flies brought up on Drosophila medium containing 6.25 %
(w/w) chili paste could survive to be adult flies; therefore, this experimental medium was
used for the investigation on both the mutagenicity and antimutagenicity of each chili paste.
In the mutagenicity evaluation, the number of induced wing spots of adult flies derived from
larvae brought up on Drosophila medium containing 6.25% chili paste was not different from
that of the flies brought up on the negative control medium (data not shown). This indicated
that most samples contain none or undetectable level of mutagens. The number of wing spots
of adult flies obtained from Drosophila medium containing both chili paste and 20 mM
urethane was compared with that of adult flies obtained from positive control group in order
to derive the modulating effect of each sample on the mutagenicity of 20 mM urethane. The
result shown in Figure 2 demonstrates that none of the pastes reduced the mutagenicity of
urethane at the significant level; the inhibitory effects (if present) of kaffir lime, kumquat,
tamarind and bilimbi pastes were less than 20% modification. On the other hand, the paste
containing lime, Madan or lemon as well as the sample base significantly potentiated the
mutagenicity of urethane; they were classified as weak or moderate potentiator. In addition,
mombin, mango and star gooseberry also could potentiate the mutagenicity at the negligible
level (less than 20% inhibition).
Figure 2. The modulation effect of each Thai chili pastes (6.25% w/w in medium) on urethane
(20 mM) induced somatic mutation and recombination in Drosophila melanogaster derived
from trans-heterozygous (mwh+/+flr3) larvae. Data are shown on either % inhibition or %
potentiation.
Discussion and Conclusion
Thai chili pastes were good sources of antioxidants including phenolic compounds.
This may be due to the fact that all pastes contained herbs namely, chili pepper (Capsicum
frutescens) and garlic (Allium sativum) as that are the sources of antioxidants [9, 10]. In
addition, each sour fruit added into Thai chili paste contains different phytochemicals
including phenolic compounds that act as antioxidants such as limonene, naringenin,
naringin, diosmin, tangeretin and rutin from citrus fruits [11]. Other fruits beside citrus
member such as mombin could make chili paste to possess the highest antioxidants and
phenolic compounds since it was identified as a source of antioxidants [12]. However, the
level of antioxidant activity and total phenolic content of sour juice was not the index of
antimutagenicity of chili paste. It was shown that mombin paste which posed the highest
antioxidant activities and total phenolic content among sour juices of this study did not inhibit
the mutagenicity of urethane while other four Thai chili pastes namely, kaffir lime paste,
kumquat paste, tamarind paste and bilimbi paste could reduce the mutagenicity of urethane at
certain levels. The information about antimutagenicity of the components of sour juices is
scarce. Most juices were characterized for their phytochemicals only on the antioxidant
activity. For example, bilimbi has tannin, saponin, oxalic acid and phenol that was reported to
have multiple biological effects, including antioxidant activity [13]; tamarind pulp extract
was rich in polyphenolic compounds that could reduce atherosclerosis progression and the
oxidative stress in hamsters and had an in vitro antioxidant effect [14]; kaffir lime contained
citronellal, geranial and d-limonene [15] which was reported to have antimicrobial,
antioxidant and cytotoxic activites [16, 17].
The present investigation suggests that high consumption of Thai chili paste should be
done with caution because the data of the present investigation suggested that it enhanced the
mutagenicity of 20 mM urethane when it was mixed with Drosophila medium. It was
proposed that when each sample was mixed with the Drosophila medium, it led to the over
consumption of some phytochemicals. It had been shown that some fruits and their processed
products namely, orange and pamelo [18], durian and mangosteen [19] increased the
mutagenicity of urethane in the somatic mutation and recombination test. The enhancing
effect of each sample might be due to some natural compounds could induce the catalytic
activities of cytochrome P-450 enzyme system (phase 1) or inhibited glutathione-Stransferase as well as decreased the amounts of glutathione of phase 2 detoxifying system in
Drosophila melanogaster [20]. Myristicin (500 µmol/kg body weight, i.p) was revealed to
cause 2-20 folds increase of liver cytochrome-P450 activities with respect to those of the
control animal [21]. Deng et al. (2007) found that quercetin (250 mg/kg body weight)
significantly increased cytochrome-P450 2E1 activity (1.31 fold) and cytochrome-P450s 2E1
protein expression (1.43 fold); however, cytochrome-P450 2E1 activity and cytochromeP450 2E1 protein expression were not significantly altered by keampferol [22]. Paolini et al.
(1999) Literature review on the effect of natural constituents of fruit on phase 2 modification
enzymes is scarce [23]. Only the works of Van Zanden et al. (2004) and Cermak (2008)
revealed that the flavonoids namely, galangin, kaempferol and quercetin and the flavones
namely, eriodyctiol had inhibitory effects on glutathione-S-transferase activity of subfamily
GSTP1-1 in transfected human MCF7 breast cancer cells. Therefore, there is a need to
determine and/or quantify the compounds that pose such activity in the samples [24, 25].
The other reason that sample base as well as some other Thai chili pastes increased
the mutagenicity of urethane might be due to shrimp paste, a salted fermented sea food.
Keawngarm and Kangsadalampai [26] investigated on the enhancing effect of four salty
foods, namely boiled salted duck egg, pickled green mussel, fried salted Spanish mackerel
and fried salted beef on urethane mutagenicity; they concluded that the enhancing effect of
such salty foods might be due to the amount of sodium chloride in the sample that could
impair the repairing system during DNA damage [27]. Vences-Mejía et al. [28] investigated
the effect of high salt on male Wistar rat and reported that feeding diets containing different
NaCl concentrations (0.6% control group, 6%, 12%, 18% and 24%) on rat for 12 weeks
caused histological changes as well as modulation the activity of cytochrome P450 in gastric
mucosa. Chronic gastritis, regenerative hyperplasia and focal metaplasia were noted in
animals receiving the 12%, 18% and 24% NaCl diets. In the same groups, induction of
CYP1A1 and CYP3A2 was produced, mainly in areas of metaplasia. The expression of
xenobiotic metabolizing enzymes in the gastric mucosa might contribute to chemical
activation in the stomach, metabolizing both exogenous and endogenous compounds
implicated in the development of gastric cancer. In addition, Yu et al. [29] suggested that
consumption of Cantonese-style salted fish for a long period of time was significantly
associated with nasopharyngeal carcinoma in Hong Kong Chinese population.
Conclusively, it is suggested that consumption of Thai chili paste is a good practice for health
concerning consumer since it is a good source of antioxidants. However, it is cautioned that high
consumption of the paste might lead to the over consumption of some antioxidants and sodium
chloride that had been proved elsewhere to potentiate the activity of some mutagens. Therefore; it
suggests that the amount of any food items consumed by consumers should be neither high nor low
but appropriate to support their good health.
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