Extended One-generation Reproductive Toxicity and Teratogenicity of Ethanol Leaf Extract of Syzygium guineense Wall. in Rats.
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Date
2021-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Addis Abeba University
Abstract
Background: Various extracts of Syzygium guineense Wall. leaves showed scientifically
corroborated effects against hypertension, diabetic mellitus, breast and colon cancers, pain,
inflammation, free radicals, snake venom, parasites, different bacterial strains, and fungi. It is
widely stated that herbal products are presumed to be safe. However, validating the efficacy and
assessing the safety of herbal products is mandatory. During pregnancy, an embryo or fetus can be
affected by exposure to a variety of chemicals. The potential teratogenicity and reproductive
toxicity of S. guineense leaf extract has not been determined yet. Therefore, the aim of this thesis
research was to investigate the teratogenicity, reproductive toxicity, developmental neurotoxicity,
and developmental immunotoxicity as well as glandular and hepato-renal toxicity of the ethanol
extract of S. guineense leaves in rats.
Methods: For the teratogenicity study, five groups of Wistar albino rats, each consisting of ten
pregnant rats were used as experimental and control animals. Groups I-III rats were treated with
250, 500, and 1000 mg/kg body weight of 70% ethanol extract of S. guineense leaves, respectively.
Groups four and five were control and ad libitum control, respectively. Rats were treated beginning
from day 6 to day 12 of gestation. Embryos and fetuses were respectively retrieved on day 12 and
day 20 of gestation. The embryos were assessed for developmental anomalies and growth
retardation. The fetuses were examined for developmental delays, growth retardation, gross
external malformations, as well as skeletal and visceral anomalies. Histopathological alterations
of the placenta also were evaluated for any treatment-related anomalies.
For the extended one-generation reproductive toxicity study, the parental Wistar rats,
20/sex/group, were randomly assigned into four groups. Groups one, two, and three received 250,
500, and 1000 mg/kg body weight of 70% ethanol extract of S. guineense leaf for 10 weeks,
respectively: two premating, two mating, three pregnancy, and three lactational weeks. In the
parental rats, the effect of extract administration on the food intake, weight gain, weight and
histology of reproductive organs, liver, kidneys, adrenal glands, and thyroid gland were evaluated.
Moreover, serum level of thyroid hormones and biochemical tests were measured. Sperm analysis
was carried out and the length of estrous cycle was measured. Reproductive indices (pre-coital
interval, pregnancy duration, mating, fertility, and gestation indices) and pregnancy outcomes also
were evaluated. Once the pregnant dams gave birth, the pups were assessed for gross anomalies at birth. The weight of pups was measured on postnatal day zero, four, seven, fourteen, and twentyone.
In addition, pups anogenital distance was measured on postnatal day four. The presence ofnipple retention was assessed on postnatal day twelve. Moreover, postnatal death of pups was
reported on postnatal day 1, 4, 7, 14, and 21. At weaning (postnatal day 21), the pups wererandomly assigned into three cohort groups: to assess reproductive toxicity (set-1), developmentalneurotoxicity (set-2), and developmental immunotoxicity (set-3). All pups assigned into the threecohort groups were orally treated on a daily basis with similar doses used for the parental rats. Set1
pups,
20/sex/group
were
treated
up
to
postnatal
day
70.
To
investigate
the
extended
effect
of
the
test
plant
on the
first-generation
rats,
the
tests conducted
on the
parental
rats
were
repeated
on theset-1
pups. In addition, weight at and the day of vaginal opening/preputial separation wererespectively evaluated in female and male set-1 rats. Set-2 pups, 10/sex/group, were treated until
postnatal day 70 and the effect of the test plant extract on the weight and histopathology of the
brain and spinal cord was investigated. Set-three pups, 10/sex/group, received the treatment untilpostnatal day 60 and the toxic effect of the plant extract on the weight and histopathology of spleen,thymus, and lymph nodes was evaluated. Data were analyzed by one-way analysis of variance and
chi-square test using SPSS version 24.
Results: The results of potential teratogenicity assessment indicated that administration of 70%ethanol extract of S. guineense leaf resulted in a significant reduction of food intake and weightgain during pregnancy in high dose treated group. It also reduced the crown-rump length, andaverage morphological score of 12 days old rat embryos. Moreover, the crown-rump length of 20days old rat fetuses was diminished by the treatment of 1000 mg/kg body weight of S. guineenseHowever, any of the doses of this plant did not produce significant effect on the number ofimplantations, resorptions, stillbirths, and live births. The external morphological and visceralexaminations of rat fetuses did not reveal any detectable structural malformations in the cranial,
nasal, and oral cavities as well as visceral organs. The ossification centers of fetal skull, vertebrae,hyoid, forelimb, and hindlimb bones were not significantly varied across all groups. However,although not statistically significant, high dose treated rat fetuses had a reduced number ofossification centers in the sternum, caudal vertebrae, metatarsal, metacarpal, and phalanges. Theweight of the fetuses and the placentae were decreased. Decidual cystic degeneration was the mostprevalent histopathological changes of the placenta of rats treated with 1000 mg/kg body weightof the test plant extract. In the extended one-generation study, the administration of S. guineense extract resulted in
significantly reduced food intake and weight gain of parental rats. Administration of 1000 mg/kg
body weight of the extract prolonged the duration of estrous cycle and pre-coital interval of female
parental rats. The mean number of litters and live births were significantly reduced in the treated
groups. Rats treated with higher doses of the plant extract also showed significantly increased
serum ALT, AST, ALP, and urea levels. Moreover, the blood glucose level of rats treated with
1000 mg/kg body weight of the extract was significantly decreased compared to that in the control
groups. The serum level of thyroid hormone (T4) was significantly reduced in the rats treated with
500 and 1000 mg/kg body weight of S. guineense extract. Treatment of the rats with the high dose
(1000 mg/kg body weight) of the plant extract significantly reduced the relative weight of the
uterus and ovaries. No significant effect was observed in the number and morphology of
spermatozoa, duration of gestation as well as mating, fertility, and gestation indices. The pup’s
weight, presence of nipple retention on male pups, anogenital distance, and number of postnatal
deaths during lactation period were not significantly varied between the treatment and control
groups. Furthermore, the weight and histopathology of reproductive organs (weight except for
uterus and ovaries), liver, kidneys, adrenal glands, and thyroid gland were not significantly
affected by treatment with S. guineense extract.
Similar to the parental rats, the result biochemical tests measured in the first-generation set-1 rats
indicated that serum levels of ALT, AST, and ALP were significantly increased while food intake,
weight gain, and serum levels of glucose and thyroid hormone were significantly decreased. In
addition, the relative weight of the seminal gland, uterus, and ovaries was reduced by treatment
with S. guineense extract (1000 mg/kg body weight). The relative weight of the other reproductive
organs, liver, kidneys, adrenal glands, and thyroid gland was not significantly affected. In the firstgeneration
rats, treatment with 1000 mg/kg body weight of S. guineense extract prolonged the
length of estrous cycle. The weight at and the day of vaginal opening/preputial separation were
not significantly altered by treatment with the test plant. Similarly, neither the relative organ
weight nor the histopathology of the brain, spinal cord, spleen, thymus, and lymph nodes was
affected by treatment with S. guineense extract.
Conclusion: In conclusion, administration of 70% ethanol leaf extracts of S. guineense resulted in
decreased food intake and weight gain of pregnant and nonpregnant rats in the high dose treatment group that indicated its toxicity at a high dose. Treatment of rats with the high dose of S. guineense
extract revealed growth and developmental delays as evidenced by reduced crown-rump length
and average morphological score of 12 days old rat embryos and lower crown-rump length of 20
days old rat fetuses as well as the average number of total and live births. The plant extracts also
affected the blood chemistry, the length of estrous cycle, and the weight of reproductive organs
that showed its toxicity at a high dose. Therefore, consumption of the plant, especially at a high
dose, may be teratogenic and toxic. Thus, regulation and monitoring of the use of S. guineense
leaves should be considered. Moreover, liberal consumption of S. guineense leaves should be taken
curiously and cautiously. Further investigation should be conducted by increasing the number of
test animals, extending the duration of treatment period, and including additional tests/organs and
other test animals.
Description
Keywords
Developmental Immunotoxicity, Developmental Neurotoxicity, Rats, Reproductive Toxicity, Syzygium guineense leaf, Teratogenicity.