PROTECTIVE EFFECTS OF WITHANIA SOMNIFERA AND …

www.wjpr.net Vol 7, Issue 13, 2018.

951

Ranjit et al. World Journal of Pharmaceutical Research

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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