STRAWBERRY FRUIT EXTRACT AMELIORATES PREGNANCY AND FETO-GESTATIONAL EFFECTS OF SODIUM FLUORIDE EXPOSURE IN MICE
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62 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 62
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
STRAWBERRY FRUIT EXTRACT AMELIORATES PREGNANCY AND
FETO-GESTATIONAL EFFECTS OF SODIUM FLUORIDE
EXPOSURE IN MICE
Khawaja Raees Ahmad,a,* Muhammad Ali Kanwal,a Kausar Raees,b Tahir Abbasc
Sargodha, Pakistan
SUMMARY: Pregnancy and feto-gestational toxicities of fluoride (F) exposure and
their amelioration on co-treatment with strawberry fruit extract were studied in
pregnant mice. There were 5 groups of 10 dams (28–30 g): control (C) [without
treatment]; F5 [given 5 ppm F-ions (from NaF) in drinking water during gestation days
(GD) 6 to 18]; F10 [10 ppm F-ions on GD 6–18]; F5S [5 ppm F-ions + 0.2 mL strawberry
fruit extract (SFE) every 12 hr on GD 6–18]; and F10S [10 ppm F-ions + 0.2mL SFE
every 12hr on GD 6–18]. The dams were euthanized on GD 18 to recover the fetuses.
At the time of recovery, each dam was examined to obtain the intact maternal body
weight, the gravid uterine weight, and the number of corpora lutea in both ovaries,
live male and female fetuses, re-absorbed implants, and dead fetuses. Highly
significant differences (p63 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 63
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
significant decline in implantation sites and the body and uterine weights of the
dams.4
Strawberry (Fragaria × ananassa) is a fruit belonging to the family Rosaceae
that possesses unique antioxidants including anthocyanines, polyphenols, and the
derivatives of ellagitannin, quercetins and ellagic acid.5 Its consumption has been
shown to result in a remarkable increase in the antioxidant capacity of blood
plasma.6 It protects against oxidative damage and has ameliorative effects against
toxicological manifestations.5,7 Previously, we have shown the toxic effects of F
exposure and their amelioration with jambul fruit pulp extract treatment in mice
testis.8 The present study, in albino Swiss Webster laboratory mice (Mus
musculus), was designed to explore the possible pregnancy and fetal toxicological
manifestations of gestational chronic (5ppm) F exposure in drinking water and the
ameliorative potentials of strawberry fruit extract (SFE) against them.
MATERIAL AND METHODS
Chemicals: Pure crystalline NaF of laboratory analytical grade was used for the
exposure of the pregnant dams.
Animal groups and housing conditions: Pregnant mice (n=50), aged 3–4 months
and weighing 28–30g, reared and nurtured in the animal house at the Department
of Biological Sciences, University of Sargodha, were used in the present study.
The Institutional and National guidelines for the care and use of laboratory
animals were strictly followed. The dams were randomly distributed in five groups
(10 in each group): control (C) [without treatment]; F5 [given 5 ppm F-ions (from
NaF) in drinking water on GD 6 to 18]; F10 [10 ppm F-ions on GD 6–18]; F5S
[5ppm F-ions + 0.2 mL SFE) every 12 hr on GD 6–18]; and F10S [10ppm F-ions +
0.2mL SFE every 12 hr on GD 6–18]. The dams placed in the control group were
given F-free drinking water. Dams in the F5 and F5S groups were provided F-free
drinking water for the first five days of gestation followed by 5 ppm F-ions in
water on GD 6–18. The females in the F10 and F10S groups were given F-free
water for the first five days of gestation followed by 10 ppm F-ions in water on
GD 6–18. The pregnant dams in the F5S and F10S groups additionally received
0.2 mL of SFE on a 12-hrly basis on GD 6–18. The optimum conditions were
22±2ºC temperature, 40–44% humidity, and 12 hr dark and light cycles. The dams
were given free access to drinking water and standard laboratory rodent diet
throughout the study.
Preparation of strawberry (Fragaria × ananassa) fruit extract (SFE): Fresh ripe
strawberry fruits, obtained from the local market, were washed in boiled cooled
(F-free) water thoroughly, air dried, and crushed in an electric blender for 15 min
to obtain a fine pulpy mass. This thick pulpy material was spun at 5000 rpm for 15
min in a centrifuge to separate the liquid supernatant from the fibrous insoluble
contents. This liquid supernatant was stored in at –30ºC in sterilized plastic
Eppendorf tubes for the animal treatments.
Preparation of sodium fluoride solution: At first, 1.125 g of NaF was dissolved
in 1000 mL of F-free drinking water (Khush Aab, PSQCA Licence No: CML/N-64 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 64
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
137/11, a product of University of Sargodha) to obtain a 500 ppm F-ions solution.
This solution was then diluted to obtain 5 and 10 ppm F solutions for future use as
drinking water for the F5, F5S, F10, and F10S animal groups on GD 6–18.
Recovery procedure and fetal data processing: On GD 18 the dams were
weighed and euthanized (cervical dislocation). The gravid uteri were completely
exposed by means of an abdominal incision and were photographed in-situ for the
gross morphological study and record (such as the number of implants and re-
absorptions, etc.). Finally, the uteri were exteriorized, washed in normal saline,
towel dried, and weighed. The uteri were then carefully opened to recover all dead,
reabsorbed, and live fetuses. The live fetuses recovered were weighed to record
the fetal mass fluctuations in each group. In each litter, the male and female fetuses
were counted separately. Both ovaries from each female were separated, placed in
70% ethanol to observe under a dissecting research binocular (Labomid CSM2) at
10 and 30×. The corpora lutea (indicative of the actual number of ovulations in
each dam) were counted in both ovaries in each case.
Statistical analyses: Data obtained for the fetal, maternal, and gestational
parameters were analyzed statistically for the level of difference between the
groups. The statistical methods employed included ANOVA (one way), Duncan’s
Multiple Range Test using SPSS software, and the χ2 test.
RESULTS
Maternal parameters: Statistical analysis (ANOVA) of the data pertaining to the
body weight of the gravid mothers indicated highly significant variations among
the groups (p65 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 65
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
Tabl e 1. Pregnancy parameters aft er exposure to fluoride, at 5 ppm and 10 ppm, and the combination of
fluoride and strawberry fruit extract (SFE) (Values are group mean ±SEM, n=10)
Parameters Groups
Control F5 F10 F5S F10S
Mean weight a b c b d
of gravid 39.88±0.58 37.81±1.33 34.33±0.88 38.22±0.31 35.64±0. 74
mothers*
Mean weight a b c d c
of gravid uteri* 16.91±0.58 12.82±0.40 9.56±0.19 14. 74±0.93 10.46±0.24
Mean litter a b c d c
weight* 13.60±0.46 9.90±0.44 6.98±0.38 11. 98±0.35 7.76±0.37
Mean fetal a b c b c
weight/group* 1.65±0.01 1.30±0. 013 0.95±0.015 1.37±0.014 0.98±0.011
Mean number
a bc c ab ab
of live 8.2±0.29 7. 6±0. 33 7.3±0.15 8.0±0.21 7.9±0.17
fetuses/group
abcd
Any t wo groups with a common s uperscript do not differ significant ly from eac h other (post hoc
analys is), *significant dif ference (p≤0.001) among the groups (ANOVA).
Table 2. Gestat ional parameters after exposure to f luoride, at 5 ppm and 10 ppm, and the com bination
of fluoride and st rawberry fruit ext ract (SFE) (n=10)
Parameters Groups
Control F5 F10 F5S F10S
Number of corpora lutea in
83 82 83 84 83
each group
Number of implants in eac h
82 81 81 82 82
group
Number of live f etuses
81 76 73 80 79
recovered in each group
Pre-implantation losses in 1 1 2 2 1
each group
Post-implantat ion loss es in
1 5 8 2 3
each group
Co-gestational losses in each
2 6 10 4 4
group
2
χ – 24.33* 82.80* 4.02 6.05
*Significant diff erence (p66 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 66
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
Fetal parameters: A similar χ2 analysis of the data relating the total number of
male and female fetuses, the number of dead fetuses, and the re-absorbed implants
in each group showed significant (p67 Research report
Fluoride 48(1)62–68
Strawberry fruit extract ameliorates pregnancy and feto-gestational 67
effects of sodium fluoride exposure in mice
January-March 2015 Ahmad, Kanwal, Raees, Abbas
scarcity of information on the topic, these results clearly support the idea that SFE
possesses a significant ameliorative potential for the F-induced pregnancy and
developmental toxicity parameters.
Strawberry (Fragaria × ananassa) has been considered an excellent source of
naturally occurring nutritional antioxidants such as vitamin C, polyphenols,
flavonoids, and anthocyanins.15,16 Thus, the potential ameliorative effect of SFE
on the pregnancy, gestational, and fetal parameters was considered to be mediated
by means of its strong anti-oxidant capacity which minimizes the F exposure
related oxidative stress on the pregnant females and their developing fetuses.
CONCLUSIONS
F ions in drinking water, at an exposure level of 5ppm or more, have been shown
to have the potential to induce pregnancy and fetal toxicity in mice. The
simultaneous consumption of SFE in pregnancy is able to mitigate these
toxicological outcomes. These findings indicate a potential nutraceutical role for
strawberry fruit juice against the possible toxic feto-gestational implications of
fluoride exposure.
ACKNOWLEDGEMENTS
The help of the University of Sargodha, Sargodha, Pakistan, for the conducting
of this research, by financial assistance and the use of laboratory and animal house
facilities, is gratefully acknowledged.
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