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Effect of Terminalia chebula fruit extract on ethylene glycol induced urolithiasis in rats
Biomedicine & Aging Pathology 2 (2012) 99–103
Available online at
www.sciencedirect.com
Original article
Effect of Terminalia chebula fruit extract on ethylene glycol induced urolithiasis in rats Anil T. Pawar ∗ , Gayatri D. Gaikwad , Kavita S. Metkari , Kiran A. Tijore , Jaydip V. Ghodasara , Bhanudas S. Kuchekar Department of Pharmacology, MAEER’s Maharashtra Institute of Pharmacy, Paud Road, Kothrud, Pune, Maharashtra 411033, India
a r t i c l e
i n f o
Article history: Received 16 May 2012 Accepted 9 July 2012 Available online 11 August 2012 Keywords: Calcium oxalate Ethylene glycol Terminalia chebula Urolithiasis
a b s t r a c t Aim: The present study was undertaken to investigate antiurolithiatic property of aqueous extract of fruit of Terminalia chebula in Wistar albino rats. Materials and methods: The protective effect of aqueous extract of Terminalia chebula fruit was evaluated at two dose levels (100 and 200 mg/kg body weight) using ethylene glycol induced calcium oxalate urolithiasis model in rats. Results: The results indicate that ethylene glycol treatment decreases calcium level in urine and increased that in the kidney tissue homogenate, which were prevented in animal receiving simultaneous treatment of extract. Extract treatment decreased the elevated levels of oxalate and phosphate in urine as well as kidney tissue homogenate. The extract supplementation also prevented the elevation of serum levels creatinine, uric acid and blood urea nitrogen. Histopathological study revealed that extract reduced histological changes and retained the normal architecture of kidney tissue. Conclusion: This finding indicates that aqueous extract of Terminalia chebula fruit possessed antiurolithiatic activity. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction Urolithiasis is a common chronic disorder in humans and the most common type of renal stone is made of calcium oxlalate [1]. Various therapies have been used to treat this complaint. The recent remedies like surgical removal, percutaneous techniques and extracorporeal shock wave lithotripsy are costly for the common man. The recurrence is quite common with these procedures and patient has to be subjected to careful follow-up for a number of years [2]. Thus, there is need for more effective alternative therapy. Medicinal plants remain an important source of new drugs. These plant products are reported to be effective in decreasing the recurrence rate of renal calculi with no side effects [3]. In the Indian traditional systems of medicine, Ayurveda, many plants including Terminalia chebula have been claimed to be useful in various complaints including urolithiasis [4,5]. Terminalia chebula (family Combretaceae) is distributed throughout the subhimalayala tracks and all deciduous forest of India. The plant has been studied for its antibacterial, antiviral, antifungal, antimutagenetic activity, antioxidant activity, hypolipidemic, adaptogenic
∗ Corresponding author. Tel.: +91 020 30273653; fax: +91 020 25460616. E-mail address: anil [email protected] (A.T. Pawar). 2210-5220/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biomag.2012.07.005
and antianaphylytic activity, gastrointestinal motility improving and anti-ulcerogenic activity, radioprotective, antidiabetic, antispasmotic, wound healing, purgative, immunomodulatory and chemopreventive activity [5]. However, so far no systematic pharmacological study has been reported to support its usefulness in urolithiasis. This study was planned to evaluate antiurolithiatic potential of aqueous fruit extract of Terminalia chebula using calcium oxalate urolithiasis rat model.
2. Materials and methods 2.1. Animals Wistar albino rats (male) weighing between 150 to 250 g were used for this study. They were procured from Agharkar Research Institute, Pune, India. The animals were allowed for acclimatization for ten days under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The animals were given standard diet supplied by Pranav Agro Industries Ltd, Sangli, India. The animals were fed regularly and water ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee (Ref. No.: MIP/IAEC/201011/M1/Appr/002).
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2.2. Chemicals Ethylene glycol was obtained from Qualigens fine chemicals, Mumbai, India. All other chemicals and reagents were of analytical grade and procured from approved chemical suppliers. 2.3. Collection and extraction of plant material Dried fruit of Terminalia chebula was purchased from local market of Pune, Maharashtra, India. It was authenticated by Dr. A. S. Upadhye, Scientist, Agharkar Research Institute, Pune, India. The coarsely powdered fruits were extracted with distilled water at 70 to 75 ◦ C for 12 h. The obtained aqueous extract of Terminalia chebula (ATC) was evaporated at 45 ◦ C and then dried in oven. The dried extract was stored in airtight container. The yield of the extract was 25.6% w/w. 2.4. Experimental design The rats were randomly divided into six groups of six animals each. Group I served as a vehicle control and maintained on regular rat food and drinking water ad libitum and received distilled water (0.5 ml/100 g p.o.). All remaining groups received calculi inducing treatment for 28 days, comprised of 0.75% v/v ethylene glycol with 1% w/v ammonium chloride in drinking water ad libitum for 3 days to accelerate lithiasis followed by only 0.75% v/v ethylene glycol for 25 days. Group II served as lithiatic control and received distilled water (0.5 ml/100 g p.o.). Groups III and IV served as preventive treatment group and received ATC at doses of 100 and 200 mg/kg respectively from 1st day to 28th day of calculi induction. Groups V and VI served as curative treatment group and received ATC at doses of 100 and 200 mg/kg respectively from 14th day to 28th day of calculi induction. The extract of Terminalia chebula was dissolved in distilled water was given once daily by oral route (0.5 ml/100 g). The two dose levels, 100 and 200 mg/kg were used to evaluate the antiurolitiatic potential of the aqueous extract of Terminalia chebula fruit against ethylene glycol induced urolithiasis in rats [6].
tissue calcium, oxalate and phosphate content. The calcium and phosphate kits (Beacon Diagnostics Pvt. Ltd., India) were used to estimate calcium and phosphate content in the kidney tissue homogenate. The oxalate level in the kidney tissue homogenate was measured according to the method of Hodgkinson [7]. The right kidney was fixed in 10% neutral buffered formalin, processed in a series of graded alcohol and xylene, embedded in paraffin wax, sectioned at 5 m and stained with H and E (Haematoxylin and Eosin) for examination under polarized light. The slides were also observed to estimate tubulointerstitial damage index [8]. 2.8. Statistical analysis All the results were expressed as mean ± SEM. The results were analyzed statistically using one-way analysis of variance (ANOVA) followed by Dunnett’s comparison test. P-values were calculated against vehicle and lithiatic control groups and P < 0.05 was considered significant. 3. Results
After urine collection of 28th day, blood was obtained from the retro-orbital sinus under anaesthetic condition and animals were sacrificed by cervical decapitation. Serum was separated by centrifugation at 10,000g for 10 min and analyzed for creatinine, uric acid and blood urea nitrogen (BUN). The creatinine kit (Span Diagnostics Ltd., India), uric acid kit (Beacon Diagnostics Pvt. Ltd., India) and BUN diagnostic kit (Crest Biosystems, India) were used to estimate serum creatinine, uric acid and BUN levels respectively.
The urine output was found to increase significantly (P < 0.05) by stone-inducing treatment. Treatment of animals by both doses of ATC (100 and 200 mg/kg) in curative as well as preventive regimen decreased urine output significantly (P < 0.05) than that of calculiinduced rats but significantly higher than that of vehicle treated rats (Table 1). Urinary calcium excretion was decreased significantly (P < 0.05) in EG-treated rats. Treatment with both doses of ATC (100 and 200 mg/kg) in only preventive regimen increased calcium excretion significantly (P < 0.05) than that of EG-treated rats (Table 1). Urinary oxalate and phosphate excretion was increased significantly (P < 0.05) in EG-treated rats. Treatment with both doses of ATC (100 and 200 mg/kg) in preventive as well as curative regimen decreased oxalate excretion significantly (P < 0.05) than that of EG-treated rats. Co-treatment with higher dose of ATC (200 mg/kg) in both preventive and curative regimen significantly (P < 0.05) decreased phosphate excretion, whereas lower dose of ATC (200 mg/kg) in only curative regimen differ phosphate excretion significantly (P < 0.05) than that of EG-treated rats (Table 1). Stone-inducing treatment to animals significantly increased of calcium, oxalate and phosphate levels in the kidney tissue homogenate. Supplementation of both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen significantly decreseaed the kidney levels of calcium, oxalate and phosphate as compared to EG-treated rats (Table 2). Serum creatinine, uric acid and BUN concentration were increased significantly (P < 0.05) in EG-treated rats as compared to vehicle control rats. Co-treatment with both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen differ the serum creatinine, uric acid and BUN concentration significantly (P < 0.05) as compared to EG-treated rats (Table 2). Kidney of EG-treated rats showed marked histological changes (Fig. 1) as well as significant (P < 0.05) increase in the TDI (Table 2), which were significantly (P < 0.05) reduced and restored near to normal by supplementation of both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen.
2.7. Kidney histopathology and homogenate analysis
4. Discussion
The abdomen was cut open to remove both kidneys from each animal. Isolated kidneys were cleaned off extraneous tissue and rinsed in ice-cold physiological saline. The left kidney was finely minced and 10% homogenate was prepared in Tris-Hcl buffer (0.02 mol/l, pH 7.4). Total kidney homogenate was used for assaying
A number of models using rats have been used to induce calcium oxalate urolithiasis [9]. The most commonly employed and simplest method is to provide ethylene glycol and ammonium chloride in drinking water. EG is readily absorbed along the intestine and is metabolized in the liver to oxalate leading to hyperoxaluria. The
2.5. Collection and analysis of urine All animals were kept in individual metabolic cages and 24 h urine samples were collected on 0, 14 and 28th day of calculi inducing treatment. After measurement of urine volume, all urine samples were analyzed for calcium, oxalate and phosphate content. Calcium and phosphate in urine were estimated using kit by Beacon Diagnostics Pvt. Ltd., India. The urine oxalate level was measured using the method of Hodgkinson [7]. 2.6. Serum analysis
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Table 1 Effect of the Terminalia chebula extract on urinary parameters in urolithiasis induced rats. Days
Group II
Group III
Group IV
Group V
Group VI
Urine volume (ml/24h) 0 5.77 ± 0.17 14 5.68 ± 0.12 28 5.75 ± 0.21
Group I
5.53 ± 0.19 6.77 ± 0.21a 10.5 ± 0.49a
5.60 ± 0.17 6.47 ± 0.21a 7.75 ± 0.37a,b
5.57 ± 0.15 6.72 ± 0.20a 8.70 ± 0.50a,b
5.57 ± 0.16 6.47 ± 0.17a 7.47 ± 0.40a,b
5.45 ± 0.14 6.40 ± 0.18a 8.32 ± 0.43a,b
Calcium (mg/24 h) 0.63 ± 0.02 0 0.62 ± 0.01 14 28 0.62 ± 0.01
0.62 ± 0.02 0.47 ± 0.03a 0.38 ± 0.02a
0.61 ± 0.02 0.58 ± 0.01 0.52 ± 0.03a,b
0.64 ± 0.02 0.63 ± 0.02 0.60 ± 0.02b
0.64 ± 0.02 0.45 ± 0.02a 0.41 ± 0.01a
0.65 ± 0.01 0.49 ± 0.02a 0.44 ± 0.01a
Oxalate (mg/24 h) 0 14 28
4.05 ± 0.22 3.92 ± 0.20 3.94 ± 0.19
4.12 ± 0.23 7.15 ± 0.31 a 13.6 ± 0.53 a
4.03 ± 0.21 5.17 ± 0.27a,b 6.86 ± 0.33a,b
3.89 ± 0.22 4.79 ± 0.25b 5.74 ± 0.28a,b
3.92 ± 0.23 7.12 ± 0.32a 11.8 ± 0.47a,b
4.04 ± 0.20 7.08 ± 0.35a 8.78 ± 0.44a,b
Phosphate (mg/24 h) 0 2.58 ± 0.17 14 2.69 ± 0.12 28 2.54 ± 0.12
2.49 ± 0.10 4.17 ± 0.11a 6.04 ± 0.17a
2.43 ± 0.14 3.30 ± 0.18a,b 4.43 ± 0.15a,b
2.66 ± 0.14 2.91 ± 0.15 b 3.70 ± 0.11a,b
2.53 ± 0.17 4.01 ± 0.11a 5.82 ± 0.15a
2.43 ± 0.13 4.02 ± 0.12a 5.07 ± 0.11a,b
P < 0.05: significant, number of animals (n) = 6, values are expressed as mean ± SEM. a Comparisons are made with Group I (Vehicle control). b Comparisons are made with Group II (Lithiatic control).
Table 2 Effect of the Terminalia chebula extract on serum and kidney parameters in urolithiasis induced rats. Parameter (unit)
Group I
Group II
Group III
Group IV
Group V
Group VI
Serum Creatinine (mg/dl) Uric acid (mg/dl) BUN (mg/dl)
0.76 ± 0.03 1.29 ± 0.11 47.37 ± 1.09
1.22 ± 0.03 a 3.13 ± 0.17a 71.48 ± 1.29a
0.83 ± 0.02b 2.04 ± 0.13a,b 54.30 ± 1.16a,b
0.79 ± 0.01b 1.80 ± 0.13a,b 51.00 ± 1.11b
1.08 ± 0.03a,b 2.56 ± 0.13a,b 64.46 ± 1.27a,b
0.89 ± 0.03a,b 2.30 ± 0.12 a,b 58.12 ± 1.18a,b
Kidney Calcium (mg/g) Oxalate (mg/g) Phosphate (mg/g)
0.31 ± 0.01 4.79 ± 0.20 3.35 ± 0.11
0.60 ± 0.02a 15.33 ± 0.38a 7.25 ± 0.16a
0.45 ± 0.01a,b 8.66 ± 0.33a,b 4.74 ± 0.13a,b
0.40 ± 0.01a,b 6.76 ± 0.29a,b 4.06 ± 0.13a,b
0.55 ± 0.01a,b 13.47 ± 0.34a,b 6.30 ± 0.14a,b
0.52 ± 0.01a,b 10.14 ± 0.31a,b 5.59 ± 0.15a,b
TDI (Arbitrary units)
0.00 ± 0.00
1.13 ± 0.13a
0.25 ± 0.16b
0.13 ± 0.13b
0.50 ± 0.27b
0.38 ± 0.18b
P < 0.05: significant, number of animals (n) = 6, values are expressed as mean ± SEM. a Comparisons are made with Group I (Vehicle control). b Comparisons are made with Group II (Lithiatic control).
oxalate precipitates in the urine as calcium oxalate as a result of its poor solubility. Calcium oxalate crystals and high oxalate levels in nephrons damages epithelial cells, inducing heterogeneous crystal nucleation and causing aggregation of crystals [10,11]. Ammonium chloride has been reported to accelerate lithiasis [12,13]. Therefore, this model was used to evaluate the antiurolithiatic potential of Terminalia chebula on calcium oxalate urolithiasis. Male rats were selected to induce urolithiasis because the urinary system of male rats resembles that of humans and earlier studies shown that the amount of stone deposition in female rats was significantly less [14]. As reported in some previous studies, increase in urine volume was observed in ethylene glycol induced urolithic rats [3]. The ATC treatment also increased urine output but less than stone-induced rats. This may be due to diuretic effect of ATC which reduces the calcium oxalate supersaturation in the urine and stone formation. Consistent with some previous reports, stone-inducing treatment caused increase in oxalate and decrease in calcium excretion in the urine [3]. The ATC treatment reduced the rate of decrease in calcium excretion. Hyperoxaluria is a risk factor in the pathogenesis of calcium oxalate stone [14]. In this study, stone induction by ethylene glycol and ammonium chloride increased urinary oxalate excretion. The reduction of oxalate excretion observed on ATC treatment. This indicates that ATC act by inhibiting some steps of oxalate synthesis from ethylene glycol. An increase in urinary phosphate excretion was observed on stone-induced rats. Previous study showed that increased urinary
phosphate excretion along with oxalate stress provide an environment appropriate for stone formation by forming calcium phosphate crystals, which induces calcium oxalate deposition [3,14]. The rate of urinary phosphate excretion was decreased by ATC treatment and thereby reduced the risk of stone formation. Calcium oxalate stone deposition decreases glomerular filtration rate due to obstruction to the flow of urine. This leads to accumulation of waste products, particularly nitrogenous substances such as creatinine, uric acid and BUN in the blood [15]. In stone-induced rats, there was significant increase in the serum levels of creatinine, uric acid and BUN. Treatment of ATC showed to prevent the elevation of serum levels of these markers. This shows that ATC act by minimizing the extent of tubular dysfunction. Consistent with some previous reports, stone induction by ethylene glycol caused an increase in the calcium, oxalate and phosphate levels in the renal tissue of urolithic rats [14]. The ATC treatment showed to prevent this increase in renal calcium, oxalate and phosphate levels. This indicates that ATC also inhibits the retention stone in the renal tubules. The urinary oxalate has been reported to cause renal tissue damage by reacting with polyunsaturated fatty acids in cell membranes [15]. Stone induction by EG and ammonium chloride caused marked histological changes and increase in tubulointerstitial damage index. The ATC treatment prevented these histological changes and restored TDI near to normal. This may be in part due to antioxidant effect of the plant [16]. In conclusion, the present investigation supports the use of the plant the plant in Folk medicine against urolithiasis. Further
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Fig. 1. Microscopic images of kidney sections of different groups (A) vehicle control, (B) urolithiatic, (C) prophylactic treatment with ATC at the dose of 100 mg/kg, (D) prophylactic treatment with ATC at the dose of 200 mg/kg, (E) curative treatment with ATC at the dose of 100 mg/kg, (F) curative treatment with ATC at the dose of 200 mg/kg, (H and E × 20).
study is needed to explore the exact active constituents of the plant and mechanism of action responsible for the antiurolithiatic activity.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors would like to acknowledge principal and management of MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facilities to carry out the study.
References [1] Doddametikurke RB, Biyani CS, Browning AJ, Cartledge JJ. The role of urinary kidney stone inhibitors and promoters in the pathogenesis of calcium containing renal stones. EAU-EBU Update Series 2007;5:126–36. [2] Prasad K, Sujatha D, Bharathi K. Herbal drugs in urolithiasis-A Review. Pharmacognosy Rev 2007;1:175–9. [3] Divakar K, Pawar AT, Chandrasekhar SB, Dighe SB, Divakar G. Protective effect of the hydro-alcoholic extract of Rubia cordifolia roots against ethylene glycol induced urolithiasis in rats. Food Chem Toxicol 2010;48:1013–8. [4] Raju D, Ilanga K, Chitra V, Ashish K. Evaluation of anti-ulcer activity of methanolic extract of Terminalia chebula fruits in experimental rats. J Pharm Sci Res 2009;1:101–7. [5] Chattopadhyay RR, Bhattacharyya SK. Terminalia chebula: An update. Pharmacognosy Rev 2007;1:151–6. [6] Maheshwar GH, Deshpande SV, Pramode HJ. Anticonvulsant activity of fruits of Terminalia chebula Retz. Against MES and PTZ induced seizures in rats. J Herb Med Toxicol 2010;4:123–6. [7] Hodgkinson A. Determination of oxalic acid in biological material. Clin Chem 1970;16:547–57.
A.T. Pawar et al. / Biomedicine & Aging Pathology 2 (2012) 99–103 [8] Rothermund L, Luckert S, Koßmehl P, Paul M, Kreutz R. Renal endothelin ETA/ETB receptor imbalance differentiates salt-sensitive from salt-resistant spontaneous hypertension. Hypertension 2001;37: 275–80. [9] Liu J, Cao Z, Zhang Z, Zhou S, Ye Z. A comparative study on several models of experimental renal calcium oxalate stones formation in rats. J Huazhong Univ Sci Technolog Med Sci 2007;27:83–7. [10] Scheid CR, Cao LC, Honeyman T, Jonassen JA. How elevated oxalate can promote kidney stone disease:changes at the surface and in the cytosol of renal cells that promote crystal adherence and growth. Front Biosci 2004;9: 797–808. [11] Thamilselvan S, khan SR, Menon M. Oxalate and calcium oxalate mediated free radical toxicity in renal epithelial cells: Effect of antioxidants. Urol Res 2003;31:3–9.
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[12] Fan J, Michael AG, Chandhoke PS. Impact of ammonium chloride administration on a rat ethylene glycol urolithiaisis model. Scanning Microsc 1999;13:299–306. [13] Atmani F, Slimani Y, Mimouni M, Hacht B. Prophylaxis of calcium oxalate stones by Herniaria hirsute on experimentally-induced nephrolithiasis in rats. BJU Int 2003;92:137–40. [14] Karadi RV, Gadge N, Alagawadi KR, Savadi RV. Effect of Moringa oleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol 2006;105:306–11. [15] Ghodasara J, Pawar A, Deshmukh C, Kuchekar B. Inhibitory effect of rutin and curcumin on experimentally-induced calcium oxalate urolithiasis in rats. Pharmacognosy Res 2010;2:388–92. [16] Ashwini R, Gajalakshmi S, Mythili S, Sathiavelu A. Teminalia chebula – A pharmacological review. J Pharm Res 2011;4:2884–7.
Available online at
www.sciencedirect.com
Original article
Effect of Terminalia chebula fruit extract on ethylene glycol induced urolithiasis in rats Anil T. Pawar ∗ , Gayatri D. Gaikwad , Kavita S. Metkari , Kiran A. Tijore , Jaydip V. Ghodasara , Bhanudas S. Kuchekar Department of Pharmacology, MAEER’s Maharashtra Institute of Pharmacy, Paud Road, Kothrud, Pune, Maharashtra 411033, India
a r t i c l e
i n f o
Article history: Received 16 May 2012 Accepted 9 July 2012 Available online 11 August 2012 Keywords: Calcium oxalate Ethylene glycol Terminalia chebula Urolithiasis
a b s t r a c t Aim: The present study was undertaken to investigate antiurolithiatic property of aqueous extract of fruit of Terminalia chebula in Wistar albino rats. Materials and methods: The protective effect of aqueous extract of Terminalia chebula fruit was evaluated at two dose levels (100 and 200 mg/kg body weight) using ethylene glycol induced calcium oxalate urolithiasis model in rats. Results: The results indicate that ethylene glycol treatment decreases calcium level in urine and increased that in the kidney tissue homogenate, which were prevented in animal receiving simultaneous treatment of extract. Extract treatment decreased the elevated levels of oxalate and phosphate in urine as well as kidney tissue homogenate. The extract supplementation also prevented the elevation of serum levels creatinine, uric acid and blood urea nitrogen. Histopathological study revealed that extract reduced histological changes and retained the normal architecture of kidney tissue. Conclusion: This finding indicates that aqueous extract of Terminalia chebula fruit possessed antiurolithiatic activity. © 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction Urolithiasis is a common chronic disorder in humans and the most common type of renal stone is made of calcium oxlalate [1]. Various therapies have been used to treat this complaint. The recent remedies like surgical removal, percutaneous techniques and extracorporeal shock wave lithotripsy are costly for the common man. The recurrence is quite common with these procedures and patient has to be subjected to careful follow-up for a number of years [2]. Thus, there is need for more effective alternative therapy. Medicinal plants remain an important source of new drugs. These plant products are reported to be effective in decreasing the recurrence rate of renal calculi with no side effects [3]. In the Indian traditional systems of medicine, Ayurveda, many plants including Terminalia chebula have been claimed to be useful in various complaints including urolithiasis [4,5]. Terminalia chebula (family Combretaceae) is distributed throughout the subhimalayala tracks and all deciduous forest of India. The plant has been studied for its antibacterial, antiviral, antifungal, antimutagenetic activity, antioxidant activity, hypolipidemic, adaptogenic
∗ Corresponding author. Tel.: +91 020 30273653; fax: +91 020 25460616. E-mail address: anil [email protected] (A.T. Pawar). 2210-5220/$ – see front matter © 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biomag.2012.07.005
and antianaphylytic activity, gastrointestinal motility improving and anti-ulcerogenic activity, radioprotective, antidiabetic, antispasmotic, wound healing, purgative, immunomodulatory and chemopreventive activity [5]. However, so far no systematic pharmacological study has been reported to support its usefulness in urolithiasis. This study was planned to evaluate antiurolithiatic potential of aqueous fruit extract of Terminalia chebula using calcium oxalate urolithiasis rat model.
2. Materials and methods 2.1. Animals Wistar albino rats (male) weighing between 150 to 250 g were used for this study. They were procured from Agharkar Research Institute, Pune, India. The animals were allowed for acclimatization for ten days under standard conditions in an animal house approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The animals were given standard diet supplied by Pranav Agro Industries Ltd, Sangli, India. The animals were fed regularly and water ad libitum. The study protocol was approved by the Institutional Animal Ethics Committee (Ref. No.: MIP/IAEC/201011/M1/Appr/002).
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2.2. Chemicals Ethylene glycol was obtained from Qualigens fine chemicals, Mumbai, India. All other chemicals and reagents were of analytical grade and procured from approved chemical suppliers. 2.3. Collection and extraction of plant material Dried fruit of Terminalia chebula was purchased from local market of Pune, Maharashtra, India. It was authenticated by Dr. A. S. Upadhye, Scientist, Agharkar Research Institute, Pune, India. The coarsely powdered fruits were extracted with distilled water at 70 to 75 ◦ C for 12 h. The obtained aqueous extract of Terminalia chebula (ATC) was evaporated at 45 ◦ C and then dried in oven. The dried extract was stored in airtight container. The yield of the extract was 25.6% w/w. 2.4. Experimental design The rats were randomly divided into six groups of six animals each. Group I served as a vehicle control and maintained on regular rat food and drinking water ad libitum and received distilled water (0.5 ml/100 g p.o.). All remaining groups received calculi inducing treatment for 28 days, comprised of 0.75% v/v ethylene glycol with 1% w/v ammonium chloride in drinking water ad libitum for 3 days to accelerate lithiasis followed by only 0.75% v/v ethylene glycol for 25 days. Group II served as lithiatic control and received distilled water (0.5 ml/100 g p.o.). Groups III and IV served as preventive treatment group and received ATC at doses of 100 and 200 mg/kg respectively from 1st day to 28th day of calculi induction. Groups V and VI served as curative treatment group and received ATC at doses of 100 and 200 mg/kg respectively from 14th day to 28th day of calculi induction. The extract of Terminalia chebula was dissolved in distilled water was given once daily by oral route (0.5 ml/100 g). The two dose levels, 100 and 200 mg/kg were used to evaluate the antiurolitiatic potential of the aqueous extract of Terminalia chebula fruit against ethylene glycol induced urolithiasis in rats [6].
tissue calcium, oxalate and phosphate content. The calcium and phosphate kits (Beacon Diagnostics Pvt. Ltd., India) were used to estimate calcium and phosphate content in the kidney tissue homogenate. The oxalate level in the kidney tissue homogenate was measured according to the method of Hodgkinson [7]. The right kidney was fixed in 10% neutral buffered formalin, processed in a series of graded alcohol and xylene, embedded in paraffin wax, sectioned at 5 m and stained with H and E (Haematoxylin and Eosin) for examination under polarized light. The slides were also observed to estimate tubulointerstitial damage index [8]. 2.8. Statistical analysis All the results were expressed as mean ± SEM. The results were analyzed statistically using one-way analysis of variance (ANOVA) followed by Dunnett’s comparison test. P-values were calculated against vehicle and lithiatic control groups and P < 0.05 was considered significant. 3. Results
After urine collection of 28th day, blood was obtained from the retro-orbital sinus under anaesthetic condition and animals were sacrificed by cervical decapitation. Serum was separated by centrifugation at 10,000g for 10 min and analyzed for creatinine, uric acid and blood urea nitrogen (BUN). The creatinine kit (Span Diagnostics Ltd., India), uric acid kit (Beacon Diagnostics Pvt. Ltd., India) and BUN diagnostic kit (Crest Biosystems, India) were used to estimate serum creatinine, uric acid and BUN levels respectively.
The urine output was found to increase significantly (P < 0.05) by stone-inducing treatment. Treatment of animals by both doses of ATC (100 and 200 mg/kg) in curative as well as preventive regimen decreased urine output significantly (P < 0.05) than that of calculiinduced rats but significantly higher than that of vehicle treated rats (Table 1). Urinary calcium excretion was decreased significantly (P < 0.05) in EG-treated rats. Treatment with both doses of ATC (100 and 200 mg/kg) in only preventive regimen increased calcium excretion significantly (P < 0.05) than that of EG-treated rats (Table 1). Urinary oxalate and phosphate excretion was increased significantly (P < 0.05) in EG-treated rats. Treatment with both doses of ATC (100 and 200 mg/kg) in preventive as well as curative regimen decreased oxalate excretion significantly (P < 0.05) than that of EG-treated rats. Co-treatment with higher dose of ATC (200 mg/kg) in both preventive and curative regimen significantly (P < 0.05) decreased phosphate excretion, whereas lower dose of ATC (200 mg/kg) in only curative regimen differ phosphate excretion significantly (P < 0.05) than that of EG-treated rats (Table 1). Stone-inducing treatment to animals significantly increased of calcium, oxalate and phosphate levels in the kidney tissue homogenate. Supplementation of both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen significantly decreseaed the kidney levels of calcium, oxalate and phosphate as compared to EG-treated rats (Table 2). Serum creatinine, uric acid and BUN concentration were increased significantly (P < 0.05) in EG-treated rats as compared to vehicle control rats. Co-treatment with both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen differ the serum creatinine, uric acid and BUN concentration significantly (P < 0.05) as compared to EG-treated rats (Table 2). Kidney of EG-treated rats showed marked histological changes (Fig. 1) as well as significant (P < 0.05) increase in the TDI (Table 2), which were significantly (P < 0.05) reduced and restored near to normal by supplementation of both doses (100 and 200 mg/kg) of ATC in both curative and preventive regimen.
2.7. Kidney histopathology and homogenate analysis
4. Discussion
The abdomen was cut open to remove both kidneys from each animal. Isolated kidneys were cleaned off extraneous tissue and rinsed in ice-cold physiological saline. The left kidney was finely minced and 10% homogenate was prepared in Tris-Hcl buffer (0.02 mol/l, pH 7.4). Total kidney homogenate was used for assaying
A number of models using rats have been used to induce calcium oxalate urolithiasis [9]. The most commonly employed and simplest method is to provide ethylene glycol and ammonium chloride in drinking water. EG is readily absorbed along the intestine and is metabolized in the liver to oxalate leading to hyperoxaluria. The
2.5. Collection and analysis of urine All animals were kept in individual metabolic cages and 24 h urine samples were collected on 0, 14 and 28th day of calculi inducing treatment. After measurement of urine volume, all urine samples were analyzed for calcium, oxalate and phosphate content. Calcium and phosphate in urine were estimated using kit by Beacon Diagnostics Pvt. Ltd., India. The urine oxalate level was measured using the method of Hodgkinson [7]. 2.6. Serum analysis
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Table 1 Effect of the Terminalia chebula extract on urinary parameters in urolithiasis induced rats. Days
Group II
Group III
Group IV
Group V
Group VI
Urine volume (ml/24h) 0 5.77 ± 0.17 14 5.68 ± 0.12 28 5.75 ± 0.21
Group I
5.53 ± 0.19 6.77 ± 0.21a 10.5 ± 0.49a
5.60 ± 0.17 6.47 ± 0.21a 7.75 ± 0.37a,b
5.57 ± 0.15 6.72 ± 0.20a 8.70 ± 0.50a,b
5.57 ± 0.16 6.47 ± 0.17a 7.47 ± 0.40a,b
5.45 ± 0.14 6.40 ± 0.18a 8.32 ± 0.43a,b
Calcium (mg/24 h) 0.63 ± 0.02 0 0.62 ± 0.01 14 28 0.62 ± 0.01
0.62 ± 0.02 0.47 ± 0.03a 0.38 ± 0.02a
0.61 ± 0.02 0.58 ± 0.01 0.52 ± 0.03a,b
0.64 ± 0.02 0.63 ± 0.02 0.60 ± 0.02b
0.64 ± 0.02 0.45 ± 0.02a 0.41 ± 0.01a
0.65 ± 0.01 0.49 ± 0.02a 0.44 ± 0.01a
Oxalate (mg/24 h) 0 14 28
4.05 ± 0.22 3.92 ± 0.20 3.94 ± 0.19
4.12 ± 0.23 7.15 ± 0.31 a 13.6 ± 0.53 a
4.03 ± 0.21 5.17 ± 0.27a,b 6.86 ± 0.33a,b
3.89 ± 0.22 4.79 ± 0.25b 5.74 ± 0.28a,b
3.92 ± 0.23 7.12 ± 0.32a 11.8 ± 0.47a,b
4.04 ± 0.20 7.08 ± 0.35a 8.78 ± 0.44a,b
Phosphate (mg/24 h) 0 2.58 ± 0.17 14 2.69 ± 0.12 28 2.54 ± 0.12
2.49 ± 0.10 4.17 ± 0.11a 6.04 ± 0.17a
2.43 ± 0.14 3.30 ± 0.18a,b 4.43 ± 0.15a,b
2.66 ± 0.14 2.91 ± 0.15 b 3.70 ± 0.11a,b
2.53 ± 0.17 4.01 ± 0.11a 5.82 ± 0.15a
2.43 ± 0.13 4.02 ± 0.12a 5.07 ± 0.11a,b
P < 0.05: significant, number of animals (n) = 6, values are expressed as mean ± SEM. a Comparisons are made with Group I (Vehicle control). b Comparisons are made with Group II (Lithiatic control).
Table 2 Effect of the Terminalia chebula extract on serum and kidney parameters in urolithiasis induced rats. Parameter (unit)
Group I
Group II
Group III
Group IV
Group V
Group VI
Serum Creatinine (mg/dl) Uric acid (mg/dl) BUN (mg/dl)
0.76 ± 0.03 1.29 ± 0.11 47.37 ± 1.09
1.22 ± 0.03 a 3.13 ± 0.17a 71.48 ± 1.29a
0.83 ± 0.02b 2.04 ± 0.13a,b 54.30 ± 1.16a,b
0.79 ± 0.01b 1.80 ± 0.13a,b 51.00 ± 1.11b
1.08 ± 0.03a,b 2.56 ± 0.13a,b 64.46 ± 1.27a,b
0.89 ± 0.03a,b 2.30 ± 0.12 a,b 58.12 ± 1.18a,b
Kidney Calcium (mg/g) Oxalate (mg/g) Phosphate (mg/g)
0.31 ± 0.01 4.79 ± 0.20 3.35 ± 0.11
0.60 ± 0.02a 15.33 ± 0.38a 7.25 ± 0.16a
0.45 ± 0.01a,b 8.66 ± 0.33a,b 4.74 ± 0.13a,b
0.40 ± 0.01a,b 6.76 ± 0.29a,b 4.06 ± 0.13a,b
0.55 ± 0.01a,b 13.47 ± 0.34a,b 6.30 ± 0.14a,b
0.52 ± 0.01a,b 10.14 ± 0.31a,b 5.59 ± 0.15a,b
TDI (Arbitrary units)
0.00 ± 0.00
1.13 ± 0.13a
0.25 ± 0.16b
0.13 ± 0.13b
0.50 ± 0.27b
0.38 ± 0.18b
P < 0.05: significant, number of animals (n) = 6, values are expressed as mean ± SEM. a Comparisons are made with Group I (Vehicle control). b Comparisons are made with Group II (Lithiatic control).
oxalate precipitates in the urine as calcium oxalate as a result of its poor solubility. Calcium oxalate crystals and high oxalate levels in nephrons damages epithelial cells, inducing heterogeneous crystal nucleation and causing aggregation of crystals [10,11]. Ammonium chloride has been reported to accelerate lithiasis [12,13]. Therefore, this model was used to evaluate the antiurolithiatic potential of Terminalia chebula on calcium oxalate urolithiasis. Male rats were selected to induce urolithiasis because the urinary system of male rats resembles that of humans and earlier studies shown that the amount of stone deposition in female rats was significantly less [14]. As reported in some previous studies, increase in urine volume was observed in ethylene glycol induced urolithic rats [3]. The ATC treatment also increased urine output but less than stone-induced rats. This may be due to diuretic effect of ATC which reduces the calcium oxalate supersaturation in the urine and stone formation. Consistent with some previous reports, stone-inducing treatment caused increase in oxalate and decrease in calcium excretion in the urine [3]. The ATC treatment reduced the rate of decrease in calcium excretion. Hyperoxaluria is a risk factor in the pathogenesis of calcium oxalate stone [14]. In this study, stone induction by ethylene glycol and ammonium chloride increased urinary oxalate excretion. The reduction of oxalate excretion observed on ATC treatment. This indicates that ATC act by inhibiting some steps of oxalate synthesis from ethylene glycol. An increase in urinary phosphate excretion was observed on stone-induced rats. Previous study showed that increased urinary
phosphate excretion along with oxalate stress provide an environment appropriate for stone formation by forming calcium phosphate crystals, which induces calcium oxalate deposition [3,14]. The rate of urinary phosphate excretion was decreased by ATC treatment and thereby reduced the risk of stone formation. Calcium oxalate stone deposition decreases glomerular filtration rate due to obstruction to the flow of urine. This leads to accumulation of waste products, particularly nitrogenous substances such as creatinine, uric acid and BUN in the blood [15]. In stone-induced rats, there was significant increase in the serum levels of creatinine, uric acid and BUN. Treatment of ATC showed to prevent the elevation of serum levels of these markers. This shows that ATC act by minimizing the extent of tubular dysfunction. Consistent with some previous reports, stone induction by ethylene glycol caused an increase in the calcium, oxalate and phosphate levels in the renal tissue of urolithic rats [14]. The ATC treatment showed to prevent this increase in renal calcium, oxalate and phosphate levels. This indicates that ATC also inhibits the retention stone in the renal tubules. The urinary oxalate has been reported to cause renal tissue damage by reacting with polyunsaturated fatty acids in cell membranes [15]. Stone induction by EG and ammonium chloride caused marked histological changes and increase in tubulointerstitial damage index. The ATC treatment prevented these histological changes and restored TDI near to normal. This may be in part due to antioxidant effect of the plant [16]. In conclusion, the present investigation supports the use of the plant the plant in Folk medicine against urolithiasis. Further
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Fig. 1. Microscopic images of kidney sections of different groups (A) vehicle control, (B) urolithiatic, (C) prophylactic treatment with ATC at the dose of 100 mg/kg, (D) prophylactic treatment with ATC at the dose of 200 mg/kg, (E) curative treatment with ATC at the dose of 100 mg/kg, (F) curative treatment with ATC at the dose of 200 mg/kg, (H and E × 20).
study is needed to explore the exact active constituents of the plant and mechanism of action responsible for the antiurolithiatic activity.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements The authors would like to acknowledge principal and management of MAEER’s Maharashtra Institute of Pharmacy, Pune for providing necessary facilities to carry out the study.
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