Antioxidant Activities, Total Phenolic Content and Antimutagenicity of Five Waffles
Consisted of Different Selected Fruit Juices.
Suchada Jaroenvong1*, Kaew Kangsadalampai#, 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: suchada.jar@student.mahidol.ac.th #e-mail: kaew.kan@mahidol.ac.th
Abstract
Fruits are natural sources of phytochemical; some are proved to have a protective
effect against cancer. If any fruit juice is incorporated into a bakery product that most people
enjoy, the protective effect against mutagens/carcinogens might be obtained. Five fruit juices
obtained from jackfruit, star gooseberry, star fruit, cashew apple and gac were prepared.
Then, a waffle fortified with a selected fruit juice was prepared and determined for
antioxidant activities, total phenolic content and antimutagenicity against urethane. The
fortified waffles had higher antioxidant activities and certain amount of phenolic compounds
than those of control waffle. The free radical (DPPH) scavenging activity ranged from 41.5
(control) to 314.8 (cashew apple) µM Trolox equivalent/100 g, the ferric reducing antioxidant
power (FRAP) ranged from 106.2 (control) to 617.3 (cashew apple) µM ferrous/100 g, and
total phenolic content ranged from 24.1 (control) to 114.4 (jackfruit) mg gallic acid
equivalent/100 g. In antimutagenicity study, the trans-heterozygous (mwh+/+flr3) Drosophila
melanogaster larvae were transferred to both an experimental medium (made by substituting
each freeze-dried fruit juice fortified waffle for corn flour and sugar) containing 20 mM
urethane and a positive (20 mM urethane) control medium. The round wings from the
surviving adult flies were analyzed for the occurrence of mutant spots and used for obtaining
the ratio between numbers of spot per wing induced by urethane in the presence and absence
of sample (MI or mutagenicity index). The difference of mutagenicity indexes seen among
samples indicated that only Jack fruit waffle had weak antimutagenicity. On the other hand,
cashew apple waffle, gac waffle and star fruit waffle enhanced the mutagenicity of urethane.
It suggested that high consumption of fruit juice fortified waffles might lead to the over
consumption of antioxidant that may potentiate the activity of some mutagens.
Keywords: Antimutagenicity, SMART, Antioxidant, Fruit juice, Waffle
Introduction
Production and consumption of animal fats and oil from plant sources have greatly
increased in recent decades, most of all in China and elsewhere in Asia. Bakery products are
energy-dense foods that are contributing to total dietary energy increases, overweight, obesity
and the risk of some cancers [1,2]. Fortunately, fruits that are natural sources of
phytochemicals such as vitamins, polyphenols and antioxidants [3,4] were proved to be
associated with a protective effect against cancer [5-7]. Flavonoids in fruits, vegetables and
juices appear to play a significant role in cancer and heart disease health benefits [8]. These
flavonoids include compounds such as quercetin, rutin, catechin and epicatechin which have
been shown in vitro to have powerful biological effects, including inhibition of eicosanoid
synthesis [9] and of platelet aggregation [10] as well as of cancer growth and development
[11]. It is hopeful that if any fruit juice is incorporated into a bakery product that most people
enjoy, the protective effect against mutagens/carcinogens might be obtained. Therefore, this
investigation was proposed to determine the antioxidant activity and antimutagenicity against
urethane in Drosophila melanogaster of the waffle containing selected fruit juice extracted
from jackfruit, star gooseberry, star fruit, cashew apple or gac.
Methodology
Chemicals and Reagents
Urethane (URE), 2, 4, 6-tripyridyl-s-triazine (TPTZ), ferric chloride hexahydrate, and
ferrous sulfate heptahydrate were purchased from Sigma Chemical (St. Louis, Mo, 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). All other chemicals and reagents were of analytical grade.
Sample Preparations
Jackfruit, star gooseberry, star fruit, cashew apple and gac were obtained from a local
market nearby Mahidol University. Each fruit was deseeded and extracted in a juice
extractor. A commercially available waffle premix (250 g), 1 egg, 180 g water or fruit juice,
25 g melted butter and 10 g milk powder were mixed and proceeded as suggested on the
package. The finished product was freeze-dried and stored frozen until used.
Antioxidant activities and total phenolic content
Each sample (1g) was extracted with 80% methanol (10 ml) at 37ºC for 1 h. The
solution was filtered through Whatman filter paper No. 1 and collected into a glass bottle.
Each methanolic extract was assayed for DPPH free radical scavenging activity, ferric
reducing antioxidant power or FRAP and the total phenolic content with modification as
suggested by Kruawan and Kangsadalampai [12].
Somatic mutation and recombination test (SMART)
Each freeze-dried waffle was 100% (w/w) replaced for corn flour and sugar in the
standard medium [13] in order to evaluate its mutagenicity. The test was performed as
described by Graf and van Schaik [14]. The standard medium containing 20 mM urethane
was used as positive control. Toxicity of each sample was determined from the data of
survival rate of adult flies. The wing spots data were evaluated using the statistical procedure
and a multiple decision procedure as described by Frei and Wurgler [15]. The presence of
each sample and 20 mM urethane in the standard medium was designed for the
antimutagenicity evaluation of the sample as suggested by Kruawan and Kangsadalampai
[12]. The modulating effect of each sample on urethane was estimated as mutagenicity index
(MI).
Spots per wing induced by urethane in the presence of sample
MI =
Spots per wing induced by urethane only
It is proposed that the MI < 0.4, 0.4-0.6, 0.6–0.8 or 0.8–1 indicates strong, moderate,
weak or negligible antimutagenicity, respectively. On the other hand, the MI higher than 1
means negligible, weak, moderate or strong potentiating effect when it is 1-1.2, 1.2-1.4, 1.41.6 or >1.6, respectively.
Results
Antioxidant activities and total phenolic content
Cashew apple juice seemed to be the best in increasing the antioxidant activities and
total phenolic contents of waffle. Star fruit and jackfruit juices were also good sources of
antioxidants and phenolic compounds. The results of all samples are shown in Figure 1. The
reducing of DPPH by each sample ranged from 41.5 to 314.8 µg Trolox equivalent
antioxidant capacity (TEAC)/100g dry weight (Figure 1A). The FRAP value of each waffle
substituted with fruit juice ranged from 106.2 to 617.3 µg Fe(II)/100g dry weight (Figure
1B). The total phenolic content of each sample ranged from 24.1 to 114.4 of mg gallic
acid/100g dry weight (Figure 1C).
TEAC
(µMTrolox/100g dry weight)
A
350
314.8
300
250
201.7
200
148.6
150
86.4
100
50
61.0
41.5
0
control
FRAP value
Fe(II)/100g dry weight.
B
Jack Fruit
Star
Cashew apple
gooseberry
700
Star fruit
Gac
617.3
600
500
376.0
356.7
400
300
200
169.8
134.7
106.2
100
0
control
GAE
(mg gallic acid/100g dry weight)
C
Jack Fruit
Star
Cashew apple
gooseberry
Star fruit
Gac
140
114.4
120
100
106.8
86.4
80
53.4
60
40
47.6
24.1
20
0
control
Jack Fruit
Star
gooseberry
Cashew
apple
Star fruit
Gac
Figure 1. Antioxidant activity in DPPH assay (A), antioxidant activity in FRAP assay (B) and total phenolic
content (C) of methanolic extracts from each waffle substituted with fruit juice
.
Mutagenicity Index (MI)
3.0
2.5
2.0
2.0
1.6
1.5
1.2
1.0
1.0
0.9
0.7
0.8
Jack Fruit
Star
gooseberry
0.5
0.0
Positive
control
control
Cashew
apple
Star fruit
Gac
Figure 2. The modulating effect of each waffle substituted with fruit juice against urethane (20 mM) induced
somatic mutation and recombination in Drosophila melanogaster derived from trans-heterozygous
(mwh+/+flr3) larvae
Antimutagenicity
Each sample was firstly tested for its mutagenicity and it was found that none was
mutagenic (data not shown). In the antimutagenicity evaluation, the difference among the
mutagenicity indexes (MI) of fortified waffles were revealed (Figure 2). Jack fruit waffle was
weak antimutagenic (MI was 0.7). Control waffle and star gooseberry waffle had no effect on
the mutagenicity of urethane (MI was between 0.8-0.9). The mutagenicity of urethane was
enhanced by cashew apple waffle (MI was 1.2), gac waffles (MI was 1.6) and star fruit
waffles (MI was 2.0).
Discussion and Conclusion
It was successful that some fruit juices could increase antioxidant activities and
phenolic compounds of waffles. Previous studies [16-22] suggested that all fruits in this study
contained various antioxidants including phenolic compounds, flavonoids, carotenoids, and
other phytochemicals; therefore, it was not surprised that fortified waffles had higher
antioxidant activities and total phenolic content than those of control waffle. Since waffle is a
bakery product and therefore the phytochemical of each juice must be heat stable in order to
express its antioxidant activities. Sridonpai [23] pasteurized (65 ºC for 30 min), boiled (100
ºC for 20 min) and sterilized (121 ºC for 15 min) cashew apple juice and found that there was
not much difference on antioxidant activities and total polyphenolic contents between original
and thermally processed one. This might explain why cashew apple juice was the best in
increasing the antioxidant activities and total phenolic contents of waffle.
The somatic mutation and recombination test (SMART) in Drosophila melanogaster
is an in vivo short term tests. It detects genetic damage in a eukaryotic organism with
metabolic machinery similar to mammalian cells [24]. It was expected that the mutagenicity
of urethane should be reduced when the testing larvae were fed the medium containing each
fortified waffle but the result came out that most samples were either ineffective or potentiate
the mutagenicity of urethane. Since each sample was substituted for all carbohydrate source
in the medium, it led to the over consumption of some natural components. Fruits and their
processed products, namely orange and pamelo [25], durian and mangosteen [26] potentiated
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 [27]. For example, the effects of the five major constituents in the
Ginkgo biloba extract, namely bilobalide, ginkogolides A, ginkogolides B, quercitin and
keampferol on the expression of the major cytochrome-P450s in rat were investigated by
Deng et al. [28]; they 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). In addition, myristicin (500 µmol/kg body weight, i.p) was an inducer of rat liver
cytochrome-P450s, namely 1A1/2, 2B1/2, and 2E1; this compound caused 2-20 folds
increase the activities of liver cytochrome-P450s with respect to those of the control animal
[29]. Literature review on the effect of natural constituents of fruit on phase 2 modification
enzymes is scarce. Only the works of Van Zanden et al. [30] and Cermak [31] 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 sample.
Conclusively, it is suggested that consumption of fortified bakery product seems to be
good practice for health concerning consumer since it was demonstrated in this investigation
that they were good sources of antioxidants. However, it is cautioned that high consumption
of fruit juice fortified waffles might lead to the over consumption of antioxidant that may
potentiate the activity of some mutagens. For instance, the consumption of high doses of
beta-carotene in the form of dietary supplement in an attempt to prevent lung and other
cancers revealed the association between the antioxidant supplement and a higher risk of lung
cancer in cigarette smokers [32]. The other demonstrated that an inappropriate amount of
antioxidant caused harmful effect; Chen et al. [33] revealed that butylatedhydroxytoluene (a
synthetic antioxidant) had little effect on the mutagenicity of 2-amino-3-methyl-imidazo[4,5f]quinoline (IQ) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) at low
concentrations, but significantly increased their mutagenicity at high concentrations.
Therefore; the present investigation has confirmed 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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