|Year : 2010 | Volume
| Issue : 1 | Page : 75-80
|Effect of extract of Withania Somnifera on dehydration-induced oxidative stress-related uremia in male rats
Koushik Das1, Tanushree Tulsian Samanta2, Pradip Samanta3, Dilip Kumar Nandi2
1 Department of Nutrition, Raja N. L. Khan Women's College, Midnapore, Dist: Paschim Medinipur, West Bengal, India
2 Department of Physiology, Raja N. L. Khan Women's College, Midnapore, Dist: Paschim Medinipur, West Bengal, India
3 Department of Microbiology, Raja N. L. Khan Women's College, Midnapore, Dist: Paschim Medinipur, West Bengal, India
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|Date of Web Publication||8-Jan-2010|
| Abstract|| |
Dehydration or water deprivation in the body decreases urinary excretion and allows urea and other protein waste products to accumulate in the blood. The aim of the present study is to evaluate the association of uremia and oxidative stress by applying the herbal plant Withania somnifera (W. somnifera) (Aswagandha). The study was performed on male Wister strain rats in which, dehydration was achieved by water withdrawal. A total of 18 rats were studied and were randomly divided into three Groups: Group-1, control, Group-2, only dehydration and Group-3, dehydration + administration of aqueous root extract of W. somnifera, orally (50 mg/100 gm body weight/day) for 25 days. After 25 days of treatment, it was observed that the body weight of Group-3 animals had increased significantly, while that in Group-2 had decreased significantly. The liver enzymes in both blood and kidneys did not show any significant change in the three groups implying absence of any toxicity of the root extract. In Group-2 animals, the serum urea and creatinine levels increased significantly when compared with animals in Groups-1 and 3. The low levels of serum urea and creatinine in Group-3 animals indicates the protective effect of the plant extract against renal injury caused by dehydration. Dehydration-induced oxidative stress was established in our study by noting the low activities of super-oxide dismutase and catalase, both important antioxidant enzymes, in Group-2 animals; both enzymes were stabilized in animals of Groups-3 and 1. In conclusion, it is hypothesized that there is an antioxidative role of W. somnifera resulting in reducing the extent of renal injury as a result of oxidative stress.
|How to cite this article:|
Das K, Samanta TT, Samanta P, Nandi DK. Effect of extract of Withania Somnifera on dehydration-induced oxidative stress-related uremia in male rats. Saudi J Kidney Dis Transpl 2010;21:75-80
|How to cite this URL:|
Das K, Samanta TT, Samanta P, Nandi DK. Effect of extract of Withania Somnifera on dehydration-induced oxidative stress-related uremia in male rats. Saudi J Kidney Dis Transpl [serial online] 2010 [cited 2020 May 26];21:75-80. Available from: http://www.sjkdt.org/text.asp?2010/21/1/75/58714
| Introduction|| |
Dehydration or water deprivation in the body decreases urinary excretion and, allows urea and other protein waste products to accumulate in the blood. Symptoms usually start with fatigue and loss of concentration.  They may include itching and muscle twitches; dry, flaky, yellowish skin; dry mouth, metallic taste, and ammonia breath; and nausea, vomiting, diarrhea, and constipation. Advanced stages affect the nervous, cardiovascular, and respiratory systems and can lead to hypertension, seizures, heart failure, and death.  If the underlying disorder cannot be treated, the resulting renal failure will require management with dialysis or kidney transplantation. One of the main causes of acute renal failure is water deprivation/dehydration.  Recent studies have shown that oxidative stress is highly prevalent in patients with renal disease.  There is good evidence indicating that uremia in general is associated with enhanced oxidative stress,  and treatment of uremic patients with hemodialysis or peritoneal dialysis has been suggested to particularly contribute to oxidative stress and reduced antioxidant levels in these patients.  The aim of this present study is to evaluate the association of renal injury and oxidative stress by applying the herbal plant Withania somnifera (W. somnifera) (Aswagandha) in dehydrationinduced uremic rats. W. somnifera is widely used in Ayurvedic medicine, the traditional medical system of India. Its height is 3-4 feet, grows as a bush, and is a member of the family Solanaceae. In India, this plant is extensively grown and at present, this plant is cultivated for medicinal purposes. Therapeutic importance of different parts of this plant has a long history and is mentioned in Charak Sanghita. Ingredients of this plant are present in many formulations prescribed for a variety of musculoskeletal disorders (e.g., arthritis, rheumatism) as well as a general tonic to increase energy, improve overall health and longevity and prevent disease in athletes, the elderly and during pregnancy.  It is also used as an antistressor and antioxidant agent.  In the present study, we aim to see if there are any antioxidative and antiuremic effects of the root of this plant.
| Materials and Methods|| |
Selection of animals and care
The study was conducted on 18 healthy, adult, male albino rats of Wister strain having a body weight of 100 ± 15 gms. They were acclimatized to laboratory conditions for two weeks prior to experimentation. Animals were housed at one rat/cage in a temperature-controlled room (22 ±2°C) with 12-12 hour dark-light cycle (8.0020.00 hours light: 20.00-8.00 hours dark) at a humidity of 50 ±10 %. They were provided with standard food and water ad libitum. Animal care was provided according to the Guiding Principle for the Care and Use of Animals. 
The root of W. somnifera was collected from Gopali, Indian Institute of Technology, Kharagpur, Paschim Medinipur district of West Bengal. The material was identified by the taxonomist of the Botany Department at the Raja N. L. Khan Women's College, Midnapore. The voucher specimens were deposited in the Department of Botany, Raja N. L. Khan Women's College.
Preparation of aqueous extract of root of W. somnifera
The plant parts were dried in an incubator for two days at 40°C, crushed in an electrical grinder and then the powder was separated. Fifty gms of powder of said plant material was extracted in 250 mL of distilled water for 18 hours in a soxhlet apparatus. Extract of the root of W. somnifera in distilled water was collected. The extract was dried at reduced pressure, stored at 0-4°C and used for the next seven days of the experiment. As per demand, extract was prepared further throughout the experimental period. When needed, the extract was suspended in deionized water and used in the study.
| Experimental Design|| |
Grouping of animals and experimental procedure
The rats were divided into three equal groups as follows:
Group-I or control Group : Six animals were assigned as the control group. They were housed at room temperature (25 ±3°C) and received normal diet and water.
Group-II or dehydration Group : Six animals were randomly placed in groups of one rat per cage with free access to dry food (Pellet diet). Dehydration was achieved by withdrawing the drinking water bottle for 24 hrs, after which 2 mL water per day was provided to each rat. This procedure was repeated for 25 consecutive days.
Group-III or dehydration with treatment by root of W. somnifera : This group also had six animals all of which had free access to dry food. Initially, these animals were subject to dehydration as in Group-II animals; additionally, they were administered aqueous extract of root of W. somnifera at the dose of 50 mg/0.5 mL deionized water/100 gm body weight/rat by forceful feeding daily at 10 AM before giving food, during the 25 days dehydration period.
Animals sacrificed and plasma and organ collection
This experimental design was continued for 25 days. After 25 days, the animals were sacrificed and blood was collected from the aorta after which the kidneys were collected for different biochemical analysis.
| Methods of Measuring the Concerned Parameters|| |
Biochemical assay of catalase activity
Catalase activity (CAT) was measured biochemically.  For the evaluation of CAT in plasma, the collected blood was centrifuged and the plasma fraction was separated. The kidneys were homogenized separately in 0.05 M Tris Hydrochloric acid (HCl) buffer solution (pH-7.0) at a tissue concentration of 50 mg/mL.
These homogenates were centrifuged separately at 10,000 g at 4°C for 10 min. Following this, 0.5 mL of hydrogen peroxide (H2O2) and 2.5 mL of distilled water were mixed and reading of absorbance was noted using a spectrophotometric cuvette at 240 nm. Forty μL of tissue supernatant and plasma were added separately and six subsequent readings were noted at 30 second intervals.
Biochemical assay of superoxide dismutase (SOD)
The kidneys were homogenized in ice-cold 100 mM Tris-cocodylate buffer to give a tissue concentration of 50 mg/mL and centrifuged at 10,000 g for 20 mins at 4°C. The SOD activity of the supernatant was estimated by measuring the percentage of inhibition of the pyragallol autooxidation by SOD.  The buffer was 50 mM Tris (pH-8.2) containing 50 mM cocodylic acid (pH-8.2), 1 mM ethylene diamine tetra acetic acid (EDTA) and 10 mM hydrochloric acid (Hcl). In a spectrophotometric cuvette, 2 mL of buffer, 100 μL of 2 mM pyragallol and 10 μL of of supernatant were poured and the absorbance was noted in spectrophotometer at 420 nm for 3 mi. One unit of SOD was defined as the enzyme activity that inhibited the autooxidation of pyragallol by 50%.
Blood Uremia Profile
Biochemical estimation of blood urea
The collected blood was centrifuged and plasma fraction was separated. Urea level of plasma was measured by commercially available standard Blood Urea Kit (Merck, Japan) by Semiautoanalyzer (Merck, Japan) by standard protocol for photometric determination of urea according to the Urease GLDH method (kinetic UV test).
Biochemical estimation of blood creatinine
The collected blood was centrifuged and plasma fraction was separated. The plasma creatinine level was measured by commercially available standard Blood Urea Kit (Merck, Japan) by Semiautoanalyzer (Merck, Japan) by standard protocol for phtotometric determination of creatinine based on Jaffe kinetic method without de-proteinization.
Biochemical estimation of Glutamate Oxaloacetate Transaminase (SGOT) and Glutamate Pyruvate Transaminase (SGPT)
For the assessment of toxicity in blood and kidney, we measured SGOT and SGPT according to the method of Goel.
| Statistical Analysis|| |
Analysis of variance (ANOVA) followed by a multiple two-tail 't' test with Bonferroni modification was used for statistical analysis of the collected data. Differences were considered significant when P< 0.05.
| Results|| |
Water intake and dehydration procedure
During the first 25 days of experimentation, Group-I animals took a maximum of 16.4 ±0.6 mL water/day/individual. Groups-II and III animals were supplied with adequate amount of food and only 2 mL water/rat/24 hrs [Table 1].
The body weight was increased at the end of the experiment in Groups-III and I when compared to their initial body weight [Table 2]. In Group-II animals, the increase in body growth was dramatically less than the other groups due to dehydration-induced oxidative stress [Table 2].
Activities of SGOT and SGPT in serum and kidney
In both blood and kidney, the levels of SGOT and SGPT were not altered significantly among the three study groups [Table 3].
The CAT activities in serum were elevated in Group-III animals when compared to Group-II [Table 4]. At the end of the experiment, the activities of this enzyme in above mentioned tissues had decreased significantly in Group-II animals when compared to Group-I [Table 4]. There was a significant protection offered to this parameter after treating the animals of the dehydration group with extract (Group-III).
Activities of SOD
Administration of extract to Group-III animals resulted in a significant elevation in the activities of SOD in serum in comparison with the animals of Groups-I and II [Table 4]. The activity of the above enzyme was significantly decreased in aforesaid tissues in Group-II when compared to Group-I [Table 4]. Treatment with the extract to animals of Group-III resulted in a significant correction of the SOD activities when compared with Group-II, and the values were similar to the control level [Table 4].
Blood urea and creatinine
The blood urea and creatinine levels were significantly increased in Group-II animals compared to Group-I animals. However, administration of extract resulted in reduction of blood urea and creatinine levels in Group-III when compared to Group-II animals [Table 5].
| Discussion|| |
Oxidative processes are inherent to life. Basic requirements such as energy production and defending the self, imply free radical generation exposing both sides of cell membranes to their potential toxic actions.  Initially, the term oxidative stress was coined to describe the biochemical imbalance between the generation of oxidants and the antioxidant defense. In contrast, the current definition underlines the importance of tissue damage resulting from the imbalance between an excessive generation of oxidant compounds and antioxidant defense mechanisms. 
Dehydration-induced oxidative stress in blood and kidney tissue was established in our study by observing the low activities of SOD and CAT, both important antioxidant enzymes, which is consistent with the observation of others.
The decrease in antioxidant enzyme activities due to dehydration might be due to their use against free radicals destruction and/or their inhibition by free radical species. 1 It is well established that SOD activity is inhibited by hydrogen peroxide that reduces Cu+2 to Cu+1 in SOD.  The reduced Cu+1 can act as promoter of hydroxyl by Haber-Weis reaction.  The reduction of hydrogen peroxide is catalyzed by CAT that protects the tissues from highly reactive hydroxyl radicals.  Reduction of hydrogen peroxide and hydro peroxides to non-toxic products are catalyzed by SOD and Catalase. 
Recent studies have shown that oxidative stress is highly prevalent in patients with renal disease.  It is known that LDL from uremic patients presents an increased susceptibility to oxidation. Uremic oxidative stress is characterized from a biochemical point of view, as a state of reactive aldehyde and oxidized thiol group accumulation, together with depletion of reduced thiol groups, which are particularly important as part of antioxidant defense. As a consequence of diminished renal catabolism and function, uremic oxidant mediators urea and creatinine, accumulate in blood.  Urea is metabolized by "salvage pathway" and transfer of urea from plasma to colon, where it is broken down to ammonia by bacterial urease. It is probable that this process occurs also in the lower ileum.  Oxidative stress appears to increase as CKD progresses and correlates significantly with level of renal function. 
In this present study, the uremic profile (creatinine and urea) showed considerably good results following treatment with the medicinal plant W. somnifera. Thus, it has been hypothesized that W. somnifera has antioxidative properties, which may prevent the progression of any kidney disorder. Additionally, the extract has no blood or renal toxic effects and can be used safely in patients with renal disease.
| Acknowledgement|| |
We thank Dr. Uday Chand Pal, Principal, Raja N. L. Khan Women's College, Midnapore, West Bengal, India for providing funding resources for this project.
Grants& Funding : Department of Physiology& Department of Nutrition, Raja N. L. Khan Women's College, Midnapore, West Bengal, India.
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Dilip Kumar Nandi
Department of Physiology, Raja N. L. Khan Women's College, Midnapore, Dist: Paschim Medinipur, Pin: 721102, West Bengal
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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