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PROTECTIVE EFFECTS OF WITHANIA SOMNIFERA AND CURCUMA
LONGA ON CALCIUM ION AND UTERUS OF ENDOSULFAN
EXPOSED MICE
Smriti Rupam1, Ranjit Kumar*
2, Arun Kumar
2 and Ashok Ghosh
2
1PG Department of Zoology, Patna University, Patna, Bihar.
2Mahavir Cancer Institute and Research Centre, Phulwarisharif, Patna, Bihar.
ABSTRACT
Agriculture has made remarkable progress in the past few decades with
the use of agrochemicals. Pesticides, fertilizers and improved seed
varieties have contributed to substantial increases in crop yields.
Despite of these benefits, pesticide uses are accompanied by potential
risks to the environment and human health. Prolonged pesticide
exposure may cause liver malfunction, immune malfunction,
neurologic impairments and reproductive effects. Medicinal plants
have been used by people of all cultures for treatment of different
diseases and toxicity since many decades. Thus the present work is
designed to study the effect of Withania somnifera and Curcuma longa
against endosulfan induced toxicity on uterus of mice. Treatment groups were administered
Endosulfan 3 mg/kg body weight by oral gavage method for 8 weeks followed by 8 weeks
administration of Withania somnifera and Curcuma longa respectively. Calcium ion levels
were increased in endosulfan administered groups with degenerated endometrium and
muscular layer, while Withania somnifera and Curcuma longa caused effective restoration in
calcium ion level and muscular layer of uterus. Thus it is concluded from the entire study that
endosulfan causes degeneration in endometrium leading to spontaneous miscarriages. It also
causes degeneration in myometrium and increase in calcium level leading to improper
muscular rhythm causing infertility in female. While Curcuma longa causes effective
restoration in calcium ion and muscular layer of uterus, Withania somnifera also causes
restoration in calcium ion and endometrium layer. Myometrium and uterine glands are also
restored effectively. Withania somnifera causes more effective restoration than Curcuma
longa on uterus of mice leading to normal fetus development and growth in uterus.
World Journal of Pharmaceutical Research SJIF Impact Factor 8.074
Volume 7, Issue 13, 951-964. Research Article ISSN 2277– 7105
Article Received on
19 May 2018,
Revised on 09 June 2018,
Accepted on 30 June 2018
DOI: 10.20959/wjpr201813-12745
*Corresponding Author
Ranjit Kumar
Mahavir Cancer Institute and
Research Centre,
Phulwarisharif, Patna, Bihar.
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KEYWORD: Calcium, Perimetrium, Myometrium, Immune malfunction, Miscarriages.
1. INTRODUCTION
Agriculture has made remarkable progress in the past few decades. World food supplies have
expanded with agrochemicals playing a major role in increasing productivity and post-harvest
crop protection. Pesticides, fertilizers and improved seed varieties have contributed to
substantial increases in crop yields (Agoramoorthy, 2008). Despite of these benefits, pesticide
uses are accompanied by potential risks to the environment and human health. Most of the
synthetic chemical pesticides are toxic to the environment, increasing soil, air and water
pollution and associated health hazards. A large number of chemicals are known or suspected
to have the potential to function as endocrine disrupting chemicals (Bergman et al., 2013).
Human health risks can result from direct exposure of farm workers to pesticides (Banerjee et
al., 2014). Prolonged pesticide exposure may cause liver malfunction, immune malfunction,
neurologic impairment, and reproductive effects (Brouwer et al., 1999). An excess mortality
from cardiovascular and respiratory diseases has been uncovered, possibly related to the
psychosocial cause in addition to the chemical contamination (Bhardwaj and Sharma, 2013).
Medicinal plants were used by people of all cultures. The Indian System of Medicine
predominantly uses plant-based raw materials for most of their preparations and formulations
to treat arthritis, rheumatism and different ailments to improve longevity (Rabe et al., 1997).
Withania somnifera is a popular herb used in traditional medicine and remedies that have
been in practice in India from ancient time. A wide range of biological activities is reported
for W. somnifera including anti-inflammatory (Khanna et al., 2007), immune modulating,
neuroprotective and anticancer activities (Rai et al., 2016). The use of Withania is suggested
in different contexts including naturopathic care for anxiety, anti-infertility care and
osteoarthritis care (Chopra et al., 2004).
The rhizome of Curcuma longa has been used in folk traditional medicine as a household
remedy for a wide range of pathological conditions such as arthritis, diarrhea and cancer
(Noorafshan et al., 2013). Curcuma extract main component, curcumin, has been shown to
exert several biological actions including anti-infectious, antithrombotic, antiatherosclerotic,
anticonvulsant and anticancer properties (Reuland et al., 2013). Curcumin, due to its
antioxidant and anti-inflammatory properties (Claramunt et al., 2009), is a multifunction
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phytochemical that can interact with multiple molecular targets, modulating cell growth,
inflammation, and apoptosis signaling pathways (Kurapati et al., 2012).
Thus the present work is designed to study the effect of Withania somnifera and Curcuma
longa against endosulfan induced toxicity on uterus of mice.
2. MATERIALS AND METHOD
Chemicals
Pesticide Endosulfan, manufactured by Excel India Pvt. Ltd., Mumbai with EC 35% was
used for the experiment. Doses of Endosulfan administered orally to the animals was 3 mg/kg
body weight.
Animals
Adult (10-12 weeks old; 30-32 gm body weight) Swiss albino female mice were housed in
groups of 6 mice per cage, maintained under controlled conditions of temperature (22 ± 2°C)
and light and provided with feed pellets (as recommended by Templeton, 1945) prepared in
the laboratory and water ad libitum.
Study Groups
The mice were set in four groups (n =6 per group) as following: The ‘control group’ received
distilled water as drinking water. The remaining three ‘treatment groups’ were administered
Endosulfan (3 mg/kg body weight) by oral gavage method for 8 weeks. After the scheduled
dosage of Endosulfan, two treated groups were administered aqueous extracts of Withania
somnifera (200 mg/kg body weight) and Curcuma longa (200 mg/kg body weight)
respectively for 8 weeks.
Sampling
The animals were sacrificed after the scheduled treatment. The mice were anesthetized with
mild ether for the purpose. The blood and tissues from the control and treated mice were
collected. Blood sample of individual mice of each group was taken in separate RIA tube by
orbital sinus puncture. After separation of the serum it was collected in separate small,
labeled vials at 4 0C for calcium ion test. The uterus from the sacrificed mice were removed
and washed three times in isotonic saline (0.85 gm/ml) and fixed for the subsequent
histological studies under light microscopy.
3. RESULTS
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Calcium ion level in the control group was 6.4 mg/dl, while after 1 week, 4 weeks and 8
weeks administration of endosulfan it gradually became 8.3 mg/dl, 8.1 mg/dl and 8.5 mg/dl.
It became 3.6 mg/dl after 4 weeks and 6.3 mg/dl after 8 weeks of Withania administration in
mice. It became 5.0 mg/dl after 4 weeks and 7.1 mg/dl after 8 weeks administration of
Curcuma. (Graph – 1).
Uterus of control mice showed normal endometrium and lumen. Myometrium were also
normal (Figure -1). Uterus of control mice showed normal transverse and longitudional
layers. Muscular layer of uterus were very well organized with normal endothelial cells and
glands (Figure -2).
Uterus of one week endosulfan administered mice showed dilated lumen and degenerated
myometrium. Many vacuolated spaces were observed in stratum basale. Clustered nuclei
were observed in surface epithelium and glandular cells (Figure -3). Uterus of four weeks
endosulfan administered mice showed clustered nuclei of surface epithelium and degenerated
muscular layer of myometrium. Many vacuolated spaces were observed in stratum basale
with more degeneration in stratum functionale. Glands were observed in dilated condition
(Figure -4). Uterus of eight weeks endosulfan administered mice showed degenerated
endometrial cells. Continuous gaps were observed in innermembrane of endometrial cells.
Degenerated cytoplasm was observed in stratum basale. Clustered nuclei were observed in
stratum functionale with severe degeneration in glands with many vacuolated spaces
(Figure -5).
Uterus of eight weeks endosulfan administered mice followed by four weeks administration
of Withania somnifera showed little bioremidial impact on stratum functionale. Stratum
basale was less restored. Glands were also observed in degeneratd condition (Figure -6).
Uterus of eight weeks endosulfan administered mice followed by eight weeks administration
of Withania showed restoration in stratum functionale to some extent. Stratum basale was
also restored to some extent (Figure -7).
Uterus of eight weeks endosulfan administered mice followed by four weeks administration
of Curcuma longa showed restoration in glands to some extent. Stratum basale was also
restored to some extent (Figure -8). Uterus of eight weeks endosulfan administered mice
followed by eight weeks administration of curcuma showed more restoration in stratum
functionale. Stratum basale showed little restoration (Figure -9).
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PHOTOPLATES
Fig. 1: Showing uterus of control mice with normal endometrium and lumen.
Myometrium were also normal. X400.
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Fig. 2: Showing uterus of control mice with normal transverse and longitudional layers.
Muscular layer of uterus were very well organized with normal endothelial cells and
glands. X300.
Fig. 3: Showing uterus of one week endosulfan administered mice with dilated lumen
and degenerated myometrium. Many vacuolated spaces were observed in stratum
basale. Clustered nuclei were observed in surface epithelium and glandular cells. X400
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Fig. 4: Showing uterus of four weeks endosulfan administered mice with clustered
nuclei of surface epithelium and degenerated muscular layer of myometrium. Many
vacuolated spaces were observed in stratum basale with severe degeneration in stratum
functionale. Glands were observed in dilated condition. X400.
Fig. 5: Showing uterus of eight weeks endosulfan administered mice with degenerated
endometrial cells. Continuous gaps were observed in innermembrane of endometrial
cells. Degenerated cytoplasm were observed in stratum basale. Clustered nuclei were
observed in stratum functionale. with severe degeneration in glands with many
vacuolated spaces. X 300.
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Fig. 6: Showing uterus of eight weeks endosulfan administered mice followed by four
weeks administration of Withania with little bioremidial impact on stratum functionale.
Stratum basale was less restored. Glands were also observed in degeneratd condition.
X300.
Fig. 7: Showing uterus of eight weeks endosulfan administered mice followed by eight
weeks administration of Withania with restoration in stratum functionale to some
extent. Stratum basale was also restored to some extent. X 300.
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Fig. 8: Showing uterus of eight weeks endosulfan administered mice followed by four
weeks administration of Curcuma with restoration in glands to some extent. Stratum
basale was also restored to some extent. X 500.
Fig. 9: Showing uterus of eight weeks endosulfan administered mice followed by eight
weeks administration of Curcuma with more restoration in stratum functionale.
Stratum basale showed little restoration. X 300.
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4. DISCUSSION
Endosulfan has been found to increase serum calcium ion concentration and deplete the
accumulation of calcium inside mitochondria causing decrease in the electromotive force
generation in mitochondria necessary for the formation of ATP. Due to least availability of
ATP, degeneration of mitochondria occurs due to toxicity, as more ATP is needed for
detoxification by Smooth Endoplasmic Reticulum (Kumar et al., 2012). In the present study,
calcium ions were increased in endosulfan administered group.
Although exposure to pesticides has been suggested as a contributing factor to a range of
women’s health disorders including infertility, polycystic ovaries, uterine fibroids and the
early onset of puberty, considerable challenges remain in attributing cause and effect (Crain
et al., 2008). Organochlorines may function as xenoestrogens and increase the risk of
hormone related cancers including breast, prostate, stomach and lung cancer (Wolff et al.,
1993). Adipose tissue can act as a depot for endocrine disrupting chemicals thereby
increasing the risk of developing endometrial cancer in obese individuals due to
bioaccumulation of lipophilic endocrine disrupting chemicals (Hardell et al., 2004). Exposure
to endocrine disrupting chemicals has been reported to be associated with early menarche
(Schoeters et al., 2008). In present study degenerated circular and longitudinal muscles were
observed. Glands of uterus showed clustered nuclei. Thick endometrium was also observed in
pesticide administered group.
Curcumin acts like an immunosuppressive phytochemical that blocks Ca+2
signaling (Kliem
et al. 2012). The uterine myocytes exhibit a phasic contractile pattern, predominantly
regulated by intracellular calcium concentration as excitation contraction coupling
(Pehlivanoğlu et al., 2013). In present study effective restoration in calcium were observed in
turmeric administered group.
Regular exposure of curcumin has been demonstrated to induce a pro-apoptotic ER stress
response in neoplastic cell lines (Bakhshi et al., 2008). A current study revealed that
curcumin suppresses the proliferation of endometrial cells by reducing E2 level through an
estrogen receptor α mediated estrogen-like effect (Zhang et al., 2013) demonstrating the
estrogenic effects of curcumin at physiological concentrations (Bachmeier et al., 2010). In a
study, curcumin has been reported to exhibit potential tocolytic effects in the rat uterus,
inhibiting normal uterine contractions in non-pregnant rats through inhibition of IP3
production and the blocking activity of oxytocin receptors in the rat myometrium (El-sayed,
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2007). In present study restoration were observed in endometrium, perimetrium as well as
muscular layer of myometrium.
In a study on male broiler chickens, supplementation of hydro alcoholic fruit extract of
Withania coagulans produced a marked increase in the serum calcium level (Hosseini et al.
2016). The extract of W. coagulans has been shown to act as calcium antagonist resembling
the effects of verapamil, a standard calcium channel blocker and relax the smooth muscles
tissues by exhibiting calcium channel blocking activity (Ali et al., 2009). In the present study,
calcium ions were restored effectively like normal group in Withania administered group.
Active principles present in the root of W. somnifera have been reported to exert powerful
antioxidant effect like anti-stress (Trivedi et al., 2017), cognition-facilitating, anti-
inflammatory and anti-aging by increasing the level of three naturally occurring antioxidant
enzymes like superoxide dismutase, catalase and glutathione peroxidase in the brain (Dhuley,
2000). W. somnifera has been suggested to be effective in arthritic syndrome producing anti-
inflammatory responses comparable to hydrocortisones (Anbalagan et al., 1984). In present
study endometrium and uterine glands were restored like normal. Longitudinal and circular
muscles were also effectively restored to normal.
5. CONCLUSIONS
Thus it is concluded from the study that endosulfan causes degeneration in endometrium
leading to spontaneous miscarriages. It also causes degeneration in myometrium and increase
in calcium ions level, leading to improper muscular rhythm causing infertility in female.
While Curcuma longa causes effective restoration in calcium ion and muscular layer of
uterus, Withania somnifera also causes restoration in calcium ion and endometrium layer.
Myometrium and uterine glands are also restored effectively. Withania somnifera causes
more effective restoration than Curcuma longa on uterus of mice leading to normal fetus
development and growth in uterus.
6. ACKNOWLEDGEMENT
The authors are grateful to Mahavir Cancer Institute and Research Centre, Patna for
providing research facilities and also thankful to research lab assistant and animal house staff
of Mahavir Cancer Institute and Research Centre for their cooperation during entire study.
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