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Anti-urolithiatic effects of Punica granatum in male rats
Journal of Ethnopharmacology 140 (2012) 234–238
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Anti-urolithiatic effects of Punica granatum in male rats N.R. Rathod a,∗ , Dipak Biswas a , H.R. Chitme b , Sanjeev Ratna c , I.S. Muchandi a , Ramesh Chandra d a
Department of Pharmacology, H.S.K College of Pharmacy, Bagalkot, Karnataka, India Oman Medical College, Muscat, Oman c Department of Biochemistry, S. Nijalingappa Medical College, Bagalkot, Karnataka, India d Dr. B.R Ambedkar Centre for Biomedical Research, New Delhi, India b
a r t i c l e
i n f o
Article history: Received 5 May 2011 Received in revised form 21 December 2011 Accepted 5 January 2012 Available online 20 January 2012 Keywords: Urolithiasis Punica granatum Ethylene glycol Cystone
a b s t r a c t Ethnopharmacological relevance: The traditional use of Punica granatum has been reported to regulate urine discharge and controls the burning sensation of urine. Materials and methods: Animals model of calcium oxalate urolithiasis was developed in male rats by adding ethylene glycol 0.75% in drinking water. The Punica granatum chloroform extract (PGCE) and Punica grantum methanol extract (PGME) orally at 100, 200 and 400 mg/kg, respectively, were administered along with ethylene glycol for 28 days. On 28 day, 24 h urine was collected from individual rats and used for estimation of urine calcium, phosphate and oxalate. The serum creatinine, urea and uric acid levels were estimated in each animal. The kidney homogenate was used for the estimation of renal oxalate contents. The paraffin kidney sections were prepared to observe the CaOx deposits. Results: The ethylene glycol control (Gr.-II) had significant (P < 0.001 vs. normal) increase in levels of urine oxalate, calcium and phosphate, serum creatinine, urea and uric acid and renal tissues oxalates, as compared to normal (Gr.-I). The paraffin kidney sections show significant histopathological changes. The treatment of PGCE and PGME at 100, 200 and 400 mg/kg doses, significantly (P < 0.001 vs. control) decreased the urine oxalate, calcium and phosphate, renal tissue oxalates and serum creatinine, urea and uric acid, in EG induced urolithiasis after 28 days. Conclusions: The PGCE and PGME at the doses of 400 mg/kg, found to be more effective in decreasing the urolithiasis and regeneration of renal tissues in male rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The urinary tract stone formation is a common disorder estimated to occur in approximately 12% of the population with a recurrence rate of 70–80% in male and 47–60% in female (Tania et al., 1999). The mechanisms involved in the formation of calcification of stone are not fully understood. It is agreed that urolithiasis involving events such as crystal nucleation, aggregation and growth of insoluble particles (Baumann, 1998). Urine is supersaturated with common stone forming minerals; however, the crystallization inhibiting capacity of urine does not allow urolithiasis in most of individual, whereas, this natural inhibition capacity is deficient in stone forming individuals (Tiselius, 2003). It has multi-factorial etiopathogenesis, involving anatomic, environmental, genetic, infections, metabolic, nutritional and socio-economic as major factors (Alessandra and Elvino, 2003). The medicinal plants have played a significant role in various ancient traditional systems of medication. Even today, plants
∗ Corresponding author. Tel.: +91 9241006244; fax: +91 8354220008. E-mail address: [email protected] (N.R. Rathod). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.003
provide a cheapest source of medicine for majority of world population, which are considered as quite safe, with minimal or no side effects (Bashir and Gilani, 2009). The World Health Organization (WHO, 2002) has also emphasized development and utilization of herbal drugs and traditional medicines for the benefit of the world population in terms of cost effectiveness and low side effect of these drugs. Today, surgical endoscopic stone removal and extracorporeal shock wave lithotripsy have revolutionized the treatment of urolithiasis but does not prevent the likelihood of new stone formation (Kalayan et al., 2009). The recurrence of stone formation is also very high (50–80%) and no suitable medical therapy is available for such stone disorders. The various therapies including thiazide as diuretic and alkali-citrate are being used to prevent the recurrence of hypercalciuria and hyperoxaluria, which induce calculi formation but evidence for their efficacy is less (Pay, 1989). In Ayurvedic medicine, the Pomegranate is considered “a Pharmacy unto itself”. The traditional use of Pomegranate seed has been reported to regulate urine discharge and controls the burning sensation of urine (Ballahb et al., 2008). In the recent decades, the extracts of leaves, seeds, fruits and flowers of
N.R. Rathod et al. / Journal of Ethnopharmacology 140 (2012) 234–238
Punica granatum have been extensively studied for potential uses including, Anti-inflammatory effects (Lee et al., 2010), antioxidant (Celik et al., 2009), protective role in atherosclerosis and thyroid dysfunctions (Singh and Kar, 2007) and cardiovascular protection (Shiraishi et al., 2002). However, no studies have so far been reported as antiurolithiatic effects of Punica granatum fruit. In the absence of any scientific evidence, an attempt was made to undertake antiurolithiatic power of Punica granatum fruit in male rats. 2. Materials and methods 2.1. Plant material The Punica granatum (PG) fruits were collected from Bijapur and Bagalkot districts, the region of North Karnataka. The plant was authenticated at Department of Botany, B.V.V. Sangha’s Science College, Bagalkot. A voucher specimen 23/2010 was deposited in the same Institute. The fruits of Punica granatum was shade-dried and uniformly powdered and subjected to hot continuous solvent extraction with petroleum ether (40–60 ◦ C) to defat, followed by chloroform and methanol extraction. The solvent was completely removed by using rotary flash evaporator and dried in lyophilizer (Mini Lyotrap, LET Scientific Ltd, UK). The percentage of extract yield PG chloroform and methanol extracts was found 3.23% and 5.31%, respectively, calculated in terms of dried weight. These dried powdered extract were formulated as suspension in distilled water using 5% Tween80 as suspending agent (Rathod et al., 2010). All the chemicals were purchased from Hi-media, Mumbai and Sigma Chemicals Co., St Louis, USA and were of analytical reagent grade. 2.2. Animals Experiments were performed in accordance with the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA). The experimental protocol in the study was approved by the Institutional Animals Ethical Committee (HSKCP/IAEC, Clear/2010–11). The male albino rats of Wistar strain, weighing 200–250 g, were obtained from central animal house of HSK College of Pharmacy, Bagalkot. The rats were housed at temperature (25 ± 1 ◦ C) with 50 ± 55% of relative humidity. Rats were fed on standard chow diet and water ad libitum. Animals were acclimatized in institutional animal house and were exposed to 12 h day and night cycle. 2.2.1. Ethylene glycol-induced urolithiasis The animals were divided into 9 groups, each group containing six animals. Group I; normal rats; received Vehicle only. Group II; (control) received ethylene glycol (EG) 0.75% in drinking water alone. Group III; received standard drugs Cystone 750 mg/kg; (Mitra et al., 1998). Groups IV–VI; fed orally with PG chloroform extract (PGCE) 100, 200 and 400 mg/kg (Asish et al., 1999), respectively, for 28 days. Groups VII–IX; fed orally with PG methanol extract (PGME) 100, 200 and 400 mg/kg (Asish et al., 1999), respectively, for 28 days. The groups II–IX, received ethylene glycol (EG) 0.75% in drinking water ad libitium for 28 days, respectively, to induce urolithiasis and generate CaOx deposition into kidneys (Christina et al., 2006).
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2.3. Biochemical parameters 2.3.1. Analysis of urine samples All the animals were kept in the individual all glass metabolic cages and the urine sample of 24 h was collected on 28th day. The urine samples were acidified by the addition of 3 N HCl, than centrifuged at 1500 rpm for 10 min using refrigerated research centrifuge (Remi centrifuge instrument, Mumbai) to remove debris and supernatant stored at −20 ◦ C, until analyzed. Urine was analyzed for oxalate (Hodgkinson and Williams, 1972), Calcium (determined by using ERBA diagnostics Mannheim GmbH, Germany) and Inorganic Phosphate contents (Phosphorous reagent kit using ERBA diagnostics Mannheim, GmbH, Germany). 2.3.2. Analysis of blood samples The 2 ml blood samples were collected by puncturing the retro orbital venous plexus from each animal in centrifuge tubes without anticoagulant and allowed to clot at room temperature. The serum was separated by centrifugation at 1500 rpm for 15 min in refrigerated research centrifuge and used for estimation of serum creatinine, urea and uric acid using commercially available kits (ERBA Diagnostic, Mannheim) and Star-21 plus semi-auto analyzer. 2.3.3. Analysis of kidney sample The animals were sacrificed under anesthesia and after dissection; both kidneys were removed and washed with cold 0.15 M KCl. The right kidney was minced with scissors and then homogenized in 0.15 M KCl, using Remi’s glass homogenizer. The homogenate was centrifuged at 1500 rpm for 10 min using refrigerated research centrifuge, to remove the cell debris. The supernatant was used for estimation of oxalates according to methods of (Hodgkinson and Williams, 1972). 2.4. Hispathological analysis The left Kidney was fixed in a 10% solution of buffered formalin (pH.7.4). The tissue was embedded in paraffin and the sections of 5 m were taken using MAC microtome (Macro scientific works, Delhi) and stained with hematoxylin–eosin. The slides were examined for renal tubular necrosis and presence of calcium oxalate crystals under binocular microscope. 3. Statistical analysis All the statistical comparison between the groups were made by means of One Way Analysis of Variance (ANOVA) and followed by Dunnett’s Multiple Comparison test. The P < 0.05 regarded as significant using, GraphPad Prism 5.01 Software (GraphPad software, San diego, CA, USA). The data expressed are Mean ± standard error of mean (S.E.M.). 4. Results The concentration of urine calcium, phosphate and oxalate present in group I–IX, were shown in (Table 1). In the present study, administration of EG of 0.75% v/v in drinking water to male rats were caused increase in calcium, phosphate and oxalate concentration in the urine of urolithiatic control (Gr.II) showing hypercalciuria, hyperphosphaturia and hyperoxaluria, respectively. The calcium, phosphate and oxalate concentration were significantly increased (P < 0.001 vs. Gr.-I) as compared to the normal (Gr.-I) as shown in (Table 1). However, treatment with PGCE (Gr.-IV–VI) and PGME (Gr.-VII–IX) at 100, 200 and 400 mg/kg, respectively, reduced significantly (P < 0.01 vs. control)
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Table 1 Effect of PGCE and PGME on urine calcium, phosphate, and oxalate in ethylene glycol induced urolithiasis in rats. SI no
Groups
Calcium (mg/dl)
1. 2. 3. 4. 5. 6. 7. 8. 9.
Normal Control Standard (Cystone) PGCE 100 mg/kg PGCE 200 mg/kg PGCE 400 mg/kg PGME 100 mg/kg PGME 200 mg/kg PGME 400 mg/kg
3.121 18.270 3.746 11.110 8.858 6.931 5.339 5.752 4.880
± ± ± ± ± ± ± ± ±
Phosphates (mg/dl)
0.676a 1.405b *** 0.525c *** 1.173c *** 0.802c *** 0.701c *** 0.495c *** 0.495c *** 1.093c ***
5.667 18.06 4.356 9.220 7.290 5.246 7.805 6.282 5.631
± ± ± ± ± ± ± ± ±
0.272a 0.440b *** 0.248c *** 0.370c *** 0.525c *** 0.206c *** 0.178c *** 0.372c *** 0.325c ***
Oxalate (mg/dl) 3.35 13.07 5.793 8.152 6.733 6.633 10.25 9.40 8.200
± ± ± ± ± ± ± ± ±
0.373a 0.381b *** 0.205c *** 0.568c *** 0.672c *** 0.249c *** 0.592c ** 0.963c *** 0.696c ***
All values are mean ± SEM (n = 6) One way analysis of variance test (ANOVA) followed by Dunnette’s multiple comparison test. PGCE, Punica granatum chloroform extract; PGME, Punica granatum Methanol extract; ‘a’ is compared with ‘b’ and ‘b’ is compared with ‘c’. ** P < 0.01 statistically significant. *** P < 0.001 statistically significant.
calcium, phosphate and oxalate excretion in urine. PGCE and PGME 400 mg/kg reduced phosphates and oxalate levels, were comparable to the Cystone treated rats (Gr.-III). Renal function was assessed by measuring serum creatinine, urea and uric acid; in normal, control and treated rats were shown in (Table 2). The serum creatinine, urea and uric acid levels were significantly (P < 0.001 vs. Gr.-I) elevated in urolithiatic control (Gr.-II) when compared with (Gr.-I) indicating renal damage. While treatment with PGCE and PGME, significantly (P < 0.001 vs. Gr.-II) reduced the levels of these NPN substances excreted by kidneys. However, PGCE and PGME 400 mg/kg significantly reversed the serum creatinine closer to standard drug Cystone values. The maximum decrease in serum creatinine was found in PGCE 400 mg/kg, showing dose dependent activity. Similarly, the serum urea and uric acid were significantly (P < 0.001 vs. Gr.-I) increased in urolithiatic control (Gr.-II) compared to normal (Gr.-I). The treatment with PGCE and PGME significantly (P < 0.001 vs. Gr.-II) decreased in serum urea and uric acid levels. In our study PGCE and PGME at 400 mg/kg, reduced the urea and uric acid levels below the normal (Gr.-I). These results indicate that the PG improves renal function in (Gr.-IV–IX) as compared to urolithiatic control (Gr.-II). The results of PG (Gr.-IV–IX) were found to be at par with the standard drug Cystone (Gr.-III). The deposition of the crystalline components namely, oxalate in the renal tissue, were significantly (P < 0.001 vs. Gr.-I) increased in the control rats (Gr.-II). Further, treatment with PGCE and PGME significantly (P < 0.001 vs. Gr.-II) reduced the renal oxalate content and other stone forming constituents in the rats of Gr.-IV–IX, were shown in (Table 2). The examination of the paraffin kidney sections (Figs. 1–9) revealed that in PGCE and PGME treated groups (Gr.-IV–IX), no crystal was found when compared to untreated group (Gr.-II). 5. Discussion The primary agents in medical management for urolithiasis, includes calcium channel blockers, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), and ␣1 adrenergic receptor antagonists. However, these treatment regimens are not free from side effects (ShekarKumaran and Patki, 2011). Therefore, alterative treatment modalities with phyto-therapeutic agents have become the mainstay of medical therapy. It was reported earlier that ethylene glycol causes hypercalciuria, hyperphosphaturia and hyperoxaluria leading to urolithiasis (Verma et al., 2009). We also find the increased levels of calcium, phosphates and oxalates in the urine of ethylene glycol treated control rats (Gr. II, Table 1). In this study, we reported that response in 28 days ethylene glycol (0.75%) v/v administration in rats; forms renal calculi composed of mainly calcium oxalate (Bahuguna et al., 2009). The mechanism for this process may be due to an increase
in the urinary concentration of oxalates. The increased urinary calcium is a factor favoring the nucleation and precipitation of calcium oxalate from urine and subsequently crystal growth (Lemann et al., 1991). According to recent reports, the pomegranate is rich in polyphenols, anthocyanins (Vidhan et al., 2010) and many alkaloids which can cause smooth muscle relaxation specifically to the urinary and billiary tract which could facilitate the expulsion of stones from both kidneys (Calixto et al., 1984). Pomegranate is a polyphenol rich fruit, which showed potential as an anti-inflammatory agent in several experimental models (Lee et al., 2010). Treatment with PGCE and PGME decreases the calcium, phosphates and oxalates levels (Gr. IV–IX, Table 1). This may be due to anti-inflammatory effect of these compounds present in PG fruit which reduces the inflammation caused by ethylene glycol induced urolithiasis. The PGCE shows much significant decrease as compared to PGME. This suggests that the active compound in PG may be lipophilic in nature which decreases the oxalate concentration. These findings were supported by histopathological studies with paraffin kidney sections. In urolithiasis, nonprotein nitrogenous (NPN) substances such as creatinine, urea and uric acid accumulate in the blood. In this study, we find that the concentration of NPN substances viz. creatinine, urea and uric acid increases in the serum of ethylene glycol treated control rats (Gr. II, Table 2). This suggests that the EG causes renal tubular damage and decreases GFR. The groups (IV–VI, Table 2) suggests that PGCE treated rats brings significant (vs. Gr. II) decrease in the urea and uric acid to the normal limit at the dose of 200 mg/kg and it further significantly decreases to below normal (Gr.-I) levels at the doses of 400 mg/kg. However, creatinine concentration decreases significantly (vs. Gr.-II), but it does not become normal. In PGME treated rats (Gr.-VII–IX, Table 2), the urea concentration becomes normal at the doses of 400 mg/kg, where as uric acid was found below normal and creatinine decreased significantly. This may be due to the muscular damage caused by oxidative stress in experimental rats. These findings suggest that the kidney function were improved in PG treated rats. The enhanced GFR may be attributed due PG’s potent antioxidant and anti-lipid per-oxidative property, as observed by (Bagri et al., 2009). Many workers have reported that Punica granatum fruit is rich in diverse classes of potentially antioxidant compounds such as -carotenes, vitamin C, vitamin E (Turk et al., 2008). Human, animal and cellular studies have shown that exposure to high levels of oxalate and CaOx crystals produce cellular injury mediated by membrane lipid per-oxidation through intracellular ROS (Reactive oxygen species) generation. The lithogenic effect caused by ethylene glycol must be mainly attributed to the oxidative damage caused by the high level of oxalate generated by EG. Therefore, a reduction in renal oxidative stress could be another effective therapeutic approach in the treatment of urolithiasis.
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Table 2 Effect of PGCE and PGME on serum creatinine, urea and uric acid and renal oxalate levels in ethylene glycol induced urolithiasis in rats. SI no
Groups
Creatinine (mg/dl)
1. 2. 3. 4. 5. 6. 7. 8. 9.
Normal Control Standard PGCE 100 mg/kg PGCE 200 mg/kg PGCE 400 mg/kg PGME 100 mg/kg PGME 200 mg/kg PGME 400 mg/kg
0.698 10.54 1.572 7.761 4.676 1.549 3.319 2.961 1.561
± ± ± ± ± ± ± ± ±
0.081a 0.324b *** 0.103c *** 0.446c *** 0.489c *** 0.198c *** 0.351c *** 0.319c *** 0.219c ***
Urea (mg/dl) 60.51 114.8 48.86 96.48 62.87 59.05 88.00 73.80 53.86
± ± ± ± ± ± ± ± ±
6.240a 1.049 b *** 2.049c *** 1.247c *** 1.934c *** 2.915c *** 1.130c *** 2.102c *** 1.853c ***
Uric acid (mg/dl) 1.989 3.639 2.696 2.628 1.426 1.108 2.608 2.657 1.397
± ± ± ± ± ± ± ± ±
0.141a 0.231b *** 0.230c * 0.296c ** 0.227c *** 0.154c *** 0.233c ** 0.076c ** 0.168c ***
Renal oxalate 9.345 18.470 9.220 9.842 8.595 7.583 11.230 10.450 8.648
± ± ± ± ± ± ± ± ±
0.284a 0.499b *** 0.436c *** 0.377c *** 0.853c *** 0.794c *** 0.483c *** 0.831c *** 0.717c ***
All value are mean ± SEM (n = 6) One way analysis of variance test (ANOVA) followed by Dunnette’s multiple comparison test. PGCE; Punica granatum chloroform extract; PGME; Punica granatum Methanol extract; ‘a’ is compared with ‘b’ and ‘b’ is compared with ‘c’. * P < 0.05 statistically significant. ** P < 0.01 statistically significant. *** P < 0.001 statistically significant.
Figs. 1–9. Fig. 1. Normal: Arrow marks indicate normal glomerular structure and Renal tubules. Fig. 2. Control: Arrow marks indicate Oxalate renal stone and tubular dilation and renal tubular damage. Fig. 3. Cystone: Arrow mark indicate little tubular dilation and normal glomeruli. Fig. 4. PGCE 100 mg/kg, arrow marks indicate Interstitial and glomerular atrophy and hemorrhage, when compare to control groups. Fig. 5. PGCE 200 mg/kg, arrow marks indicate tubular dilation and Bowmen’s capsule space dilated and partial atrophy of glomeruli. Fig. 6. PGCE 400 mg/kg, arrow marks indicate little tubular dilation and normal glomeruli. Fig. 7. PGME 100 mg/kg, arrow mark indicate Bowmen’s capsule space with tubular dilation. Fig. 8. PGME 200 mg/kg, arrow mark indicate partial atrophy of glomeruli little tubular dilation and normal glomeruli. Fig. 9. PGME 400 mg/kg, arrow mark indicate little tubular dilation and normal glomeruli.
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Recently, it was shown that the effects of Punica granatum juice on hyperoxaluria-induced in the rats kidney, shown that the inhibitory effects on renal tubular cell injury and oxidative stress caused by oxalate crystals by reducing ROS, iNOS (Inducible nitric oxide synthase) and nuclear factor-kappaB expression (Tugcu et al., 2008). The decreased concentration of creatinine, urea and uric acid in serum and calcium, phosphate and oxalate in urine and no oxalates deposited in renal tissue, in PGCE and PGME treated rats were attributed to rich antioxidants present in these extracts. Vitamin E may prevent calcium oxalate crystal deposition in the kidney by preventing hyperoxaluria-induced per-oxidative damage to the renal tubular membrane surface, which in turned can prevent calcium oxalate crystal attachment and subsequent development of the kidney stones (Touhami et al., 2007). 6. Conclusion The administration of PGCE and PGME with EG induced urolithiatic rats resulted in removal of deposition of CaOx crystals into kidneys, improving renal histology and GFR. The observed action of the PG may be due to the prevention of urinary supersaturation, inhibition of mineralization of stone-forming constituents, normalization of the cellular function by neutralizing the effect of ROS, which could have caused oxidative stress in renal tubules. The study suggest that antioxidants, polyphenols and alkaloid of PG is therapeutically effective for the treatment of calcium oxalate stones, exhibiting effects through a combination of antioxidant and anti-inflammatory action, which could be responsible for its antilithiatic activity. Further, control studies are necessary to know the mechanism of action of PG in treatment of urolithiasis. Acknowledgements The authors are extremely thankful to Dr. V.D. Domble, Prof. & HOD of Pathology department, S.N Medical College, Bagalkot for his valuable comments on histopathological study and M/S Himalaya drug company for providing free drug sample of Cystone for this study. References Alessandra, C.P., Elvino, J.G.B., 2003. Dietary calcium intake among patients with urinary calculi. Nutritional Research 23, 1651–1660. Asish, K.D., Subhash, C.M., Sanjay, K.B., Sanghamitra, S., Das, J., Saha, B.P., Pal, M., 1999. Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. Journal of Ethnopharmacology 68, 205–208. Bagri, P., Ali, M., Aeri, V., Bhowmik, M., Sultana, S., 2009. Antidiabetic effect of Punica granatum flowers: effect on hyperlipidemia, pancreatic cell lipid peroxidation and antioxidant enzymes in experimental diabetes. Food and Chemical Toxicology 47, 50–54. Bahuguna, Y., Rawat, M.S.M., Juyal, V., Gupta, V., 2009. Antilithiatic effect of flower of Jasminum Auriculatun Vahl. International Journal of Green Pharmacy 3, 155–158. Ballahb, B., Chaurasia, O.P., Ahmed, Z., Singh, S.B., 2008. Traditional medicinal plants of cold desert Ladakh-used against kidney and urinary disorders. Journal of Ethnopharmacology 118, 331–339.
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Anti-urolithiatic effects of Punica granatum in male rats N.R. Rathod a,∗ , Dipak Biswas a , H.R. Chitme b , Sanjeev Ratna c , I.S. Muchandi a , Ramesh Chandra d a
Department of Pharmacology, H.S.K College of Pharmacy, Bagalkot, Karnataka, India Oman Medical College, Muscat, Oman c Department of Biochemistry, S. Nijalingappa Medical College, Bagalkot, Karnataka, India d Dr. B.R Ambedkar Centre for Biomedical Research, New Delhi, India b
a r t i c l e
i n f o
Article history: Received 5 May 2011 Received in revised form 21 December 2011 Accepted 5 January 2012 Available online 20 January 2012 Keywords: Urolithiasis Punica granatum Ethylene glycol Cystone
a b s t r a c t Ethnopharmacological relevance: The traditional use of Punica granatum has been reported to regulate urine discharge and controls the burning sensation of urine. Materials and methods: Animals model of calcium oxalate urolithiasis was developed in male rats by adding ethylene glycol 0.75% in drinking water. The Punica granatum chloroform extract (PGCE) and Punica grantum methanol extract (PGME) orally at 100, 200 and 400 mg/kg, respectively, were administered along with ethylene glycol for 28 days. On 28 day, 24 h urine was collected from individual rats and used for estimation of urine calcium, phosphate and oxalate. The serum creatinine, urea and uric acid levels were estimated in each animal. The kidney homogenate was used for the estimation of renal oxalate contents. The paraffin kidney sections were prepared to observe the CaOx deposits. Results: The ethylene glycol control (Gr.-II) had significant (P < 0.001 vs. normal) increase in levels of urine oxalate, calcium and phosphate, serum creatinine, urea and uric acid and renal tissues oxalates, as compared to normal (Gr.-I). The paraffin kidney sections show significant histopathological changes. The treatment of PGCE and PGME at 100, 200 and 400 mg/kg doses, significantly (P < 0.001 vs. control) decreased the urine oxalate, calcium and phosphate, renal tissue oxalates and serum creatinine, urea and uric acid, in EG induced urolithiasis after 28 days. Conclusions: The PGCE and PGME at the doses of 400 mg/kg, found to be more effective in decreasing the urolithiasis and regeneration of renal tissues in male rats. © 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The urinary tract stone formation is a common disorder estimated to occur in approximately 12% of the population with a recurrence rate of 70–80% in male and 47–60% in female (Tania et al., 1999). The mechanisms involved in the formation of calcification of stone are not fully understood. It is agreed that urolithiasis involving events such as crystal nucleation, aggregation and growth of insoluble particles (Baumann, 1998). Urine is supersaturated with common stone forming minerals; however, the crystallization inhibiting capacity of urine does not allow urolithiasis in most of individual, whereas, this natural inhibition capacity is deficient in stone forming individuals (Tiselius, 2003). It has multi-factorial etiopathogenesis, involving anatomic, environmental, genetic, infections, metabolic, nutritional and socio-economic as major factors (Alessandra and Elvino, 2003). The medicinal plants have played a significant role in various ancient traditional systems of medication. Even today, plants
∗ Corresponding author. Tel.: +91 9241006244; fax: +91 8354220008. E-mail address: [email protected] (N.R. Rathod). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.003
provide a cheapest source of medicine for majority of world population, which are considered as quite safe, with minimal or no side effects (Bashir and Gilani, 2009). The World Health Organization (WHO, 2002) has also emphasized development and utilization of herbal drugs and traditional medicines for the benefit of the world population in terms of cost effectiveness and low side effect of these drugs. Today, surgical endoscopic stone removal and extracorporeal shock wave lithotripsy have revolutionized the treatment of urolithiasis but does not prevent the likelihood of new stone formation (Kalayan et al., 2009). The recurrence of stone formation is also very high (50–80%) and no suitable medical therapy is available for such stone disorders. The various therapies including thiazide as diuretic and alkali-citrate are being used to prevent the recurrence of hypercalciuria and hyperoxaluria, which induce calculi formation but evidence for their efficacy is less (Pay, 1989). In Ayurvedic medicine, the Pomegranate is considered “a Pharmacy unto itself”. The traditional use of Pomegranate seed has been reported to regulate urine discharge and controls the burning sensation of urine (Ballahb et al., 2008). In the recent decades, the extracts of leaves, seeds, fruits and flowers of
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Punica granatum have been extensively studied for potential uses including, Anti-inflammatory effects (Lee et al., 2010), antioxidant (Celik et al., 2009), protective role in atherosclerosis and thyroid dysfunctions (Singh and Kar, 2007) and cardiovascular protection (Shiraishi et al., 2002). However, no studies have so far been reported as antiurolithiatic effects of Punica granatum fruit. In the absence of any scientific evidence, an attempt was made to undertake antiurolithiatic power of Punica granatum fruit in male rats. 2. Materials and methods 2.1. Plant material The Punica granatum (PG) fruits were collected from Bijapur and Bagalkot districts, the region of North Karnataka. The plant was authenticated at Department of Botany, B.V.V. Sangha’s Science College, Bagalkot. A voucher specimen 23/2010 was deposited in the same Institute. The fruits of Punica granatum was shade-dried and uniformly powdered and subjected to hot continuous solvent extraction with petroleum ether (40–60 ◦ C) to defat, followed by chloroform and methanol extraction. The solvent was completely removed by using rotary flash evaporator and dried in lyophilizer (Mini Lyotrap, LET Scientific Ltd, UK). The percentage of extract yield PG chloroform and methanol extracts was found 3.23% and 5.31%, respectively, calculated in terms of dried weight. These dried powdered extract were formulated as suspension in distilled water using 5% Tween80 as suspending agent (Rathod et al., 2010). All the chemicals were purchased from Hi-media, Mumbai and Sigma Chemicals Co., St Louis, USA and were of analytical reagent grade. 2.2. Animals Experiments were performed in accordance with the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA). The experimental protocol in the study was approved by the Institutional Animals Ethical Committee (HSKCP/IAEC, Clear/2010–11). The male albino rats of Wistar strain, weighing 200–250 g, were obtained from central animal house of HSK College of Pharmacy, Bagalkot. The rats were housed at temperature (25 ± 1 ◦ C) with 50 ± 55% of relative humidity. Rats were fed on standard chow diet and water ad libitum. Animals were acclimatized in institutional animal house and were exposed to 12 h day and night cycle. 2.2.1. Ethylene glycol-induced urolithiasis The animals were divided into 9 groups, each group containing six animals. Group I; normal rats; received Vehicle only. Group II; (control) received ethylene glycol (EG) 0.75% in drinking water alone. Group III; received standard drugs Cystone 750 mg/kg; (Mitra et al., 1998). Groups IV–VI; fed orally with PG chloroform extract (PGCE) 100, 200 and 400 mg/kg (Asish et al., 1999), respectively, for 28 days. Groups VII–IX; fed orally with PG methanol extract (PGME) 100, 200 and 400 mg/kg (Asish et al., 1999), respectively, for 28 days. The groups II–IX, received ethylene glycol (EG) 0.75% in drinking water ad libitium for 28 days, respectively, to induce urolithiasis and generate CaOx deposition into kidneys (Christina et al., 2006).
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2.3. Biochemical parameters 2.3.1. Analysis of urine samples All the animals were kept in the individual all glass metabolic cages and the urine sample of 24 h was collected on 28th day. The urine samples were acidified by the addition of 3 N HCl, than centrifuged at 1500 rpm for 10 min using refrigerated research centrifuge (Remi centrifuge instrument, Mumbai) to remove debris and supernatant stored at −20 ◦ C, until analyzed. Urine was analyzed for oxalate (Hodgkinson and Williams, 1972), Calcium (determined by using ERBA diagnostics Mannheim GmbH, Germany) and Inorganic Phosphate contents (Phosphorous reagent kit using ERBA diagnostics Mannheim, GmbH, Germany). 2.3.2. Analysis of blood samples The 2 ml blood samples were collected by puncturing the retro orbital venous plexus from each animal in centrifuge tubes without anticoagulant and allowed to clot at room temperature. The serum was separated by centrifugation at 1500 rpm for 15 min in refrigerated research centrifuge and used for estimation of serum creatinine, urea and uric acid using commercially available kits (ERBA Diagnostic, Mannheim) and Star-21 plus semi-auto analyzer. 2.3.3. Analysis of kidney sample The animals were sacrificed under anesthesia and after dissection; both kidneys were removed and washed with cold 0.15 M KCl. The right kidney was minced with scissors and then homogenized in 0.15 M KCl, using Remi’s glass homogenizer. The homogenate was centrifuged at 1500 rpm for 10 min using refrigerated research centrifuge, to remove the cell debris. The supernatant was used for estimation of oxalates according to methods of (Hodgkinson and Williams, 1972). 2.4. Hispathological analysis The left Kidney was fixed in a 10% solution of buffered formalin (pH.7.4). The tissue was embedded in paraffin and the sections of 5 m were taken using MAC microtome (Macro scientific works, Delhi) and stained with hematoxylin–eosin. The slides were examined for renal tubular necrosis and presence of calcium oxalate crystals under binocular microscope. 3. Statistical analysis All the statistical comparison between the groups were made by means of One Way Analysis of Variance (ANOVA) and followed by Dunnett’s Multiple Comparison test. The P < 0.05 regarded as significant using, GraphPad Prism 5.01 Software (GraphPad software, San diego, CA, USA). The data expressed are Mean ± standard error of mean (S.E.M.). 4. Results The concentration of urine calcium, phosphate and oxalate present in group I–IX, were shown in (Table 1). In the present study, administration of EG of 0.75% v/v in drinking water to male rats were caused increase in calcium, phosphate and oxalate concentration in the urine of urolithiatic control (Gr.II) showing hypercalciuria, hyperphosphaturia and hyperoxaluria, respectively. The calcium, phosphate and oxalate concentration were significantly increased (P < 0.001 vs. Gr.-I) as compared to the normal (Gr.-I) as shown in (Table 1). However, treatment with PGCE (Gr.-IV–VI) and PGME (Gr.-VII–IX) at 100, 200 and 400 mg/kg, respectively, reduced significantly (P < 0.01 vs. control)
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Table 1 Effect of PGCE and PGME on urine calcium, phosphate, and oxalate in ethylene glycol induced urolithiasis in rats. SI no
Groups
Calcium (mg/dl)
1. 2. 3. 4. 5. 6. 7. 8. 9.
Normal Control Standard (Cystone) PGCE 100 mg/kg PGCE 200 mg/kg PGCE 400 mg/kg PGME 100 mg/kg PGME 200 mg/kg PGME 400 mg/kg
3.121 18.270 3.746 11.110 8.858 6.931 5.339 5.752 4.880
± ± ± ± ± ± ± ± ±
Phosphates (mg/dl)
0.676a 1.405b *** 0.525c *** 1.173c *** 0.802c *** 0.701c *** 0.495c *** 0.495c *** 1.093c ***
5.667 18.06 4.356 9.220 7.290 5.246 7.805 6.282 5.631
± ± ± ± ± ± ± ± ±
0.272a 0.440b *** 0.248c *** 0.370c *** 0.525c *** 0.206c *** 0.178c *** 0.372c *** 0.325c ***
Oxalate (mg/dl) 3.35 13.07 5.793 8.152 6.733 6.633 10.25 9.40 8.200
± ± ± ± ± ± ± ± ±
0.373a 0.381b *** 0.205c *** 0.568c *** 0.672c *** 0.249c *** 0.592c ** 0.963c *** 0.696c ***
All values are mean ± SEM (n = 6) One way analysis of variance test (ANOVA) followed by Dunnette’s multiple comparison test. PGCE, Punica granatum chloroform extract; PGME, Punica granatum Methanol extract; ‘a’ is compared with ‘b’ and ‘b’ is compared with ‘c’. ** P < 0.01 statistically significant. *** P < 0.001 statistically significant.
calcium, phosphate and oxalate excretion in urine. PGCE and PGME 400 mg/kg reduced phosphates and oxalate levels, were comparable to the Cystone treated rats (Gr.-III). Renal function was assessed by measuring serum creatinine, urea and uric acid; in normal, control and treated rats were shown in (Table 2). The serum creatinine, urea and uric acid levels were significantly (P < 0.001 vs. Gr.-I) elevated in urolithiatic control (Gr.-II) when compared with (Gr.-I) indicating renal damage. While treatment with PGCE and PGME, significantly (P < 0.001 vs. Gr.-II) reduced the levels of these NPN substances excreted by kidneys. However, PGCE and PGME 400 mg/kg significantly reversed the serum creatinine closer to standard drug Cystone values. The maximum decrease in serum creatinine was found in PGCE 400 mg/kg, showing dose dependent activity. Similarly, the serum urea and uric acid were significantly (P < 0.001 vs. Gr.-I) increased in urolithiatic control (Gr.-II) compared to normal (Gr.-I). The treatment with PGCE and PGME significantly (P < 0.001 vs. Gr.-II) decreased in serum urea and uric acid levels. In our study PGCE and PGME at 400 mg/kg, reduced the urea and uric acid levels below the normal (Gr.-I). These results indicate that the PG improves renal function in (Gr.-IV–IX) as compared to urolithiatic control (Gr.-II). The results of PG (Gr.-IV–IX) were found to be at par with the standard drug Cystone (Gr.-III). The deposition of the crystalline components namely, oxalate in the renal tissue, were significantly (P < 0.001 vs. Gr.-I) increased in the control rats (Gr.-II). Further, treatment with PGCE and PGME significantly (P < 0.001 vs. Gr.-II) reduced the renal oxalate content and other stone forming constituents in the rats of Gr.-IV–IX, were shown in (Table 2). The examination of the paraffin kidney sections (Figs. 1–9) revealed that in PGCE and PGME treated groups (Gr.-IV–IX), no crystal was found when compared to untreated group (Gr.-II). 5. Discussion The primary agents in medical management for urolithiasis, includes calcium channel blockers, steroids, non-steroidal anti-inflammatory drugs (NSAIDs), and ␣1 adrenergic receptor antagonists. However, these treatment regimens are not free from side effects (ShekarKumaran and Patki, 2011). Therefore, alterative treatment modalities with phyto-therapeutic agents have become the mainstay of medical therapy. It was reported earlier that ethylene glycol causes hypercalciuria, hyperphosphaturia and hyperoxaluria leading to urolithiasis (Verma et al., 2009). We also find the increased levels of calcium, phosphates and oxalates in the urine of ethylene glycol treated control rats (Gr. II, Table 1). In this study, we reported that response in 28 days ethylene glycol (0.75%) v/v administration in rats; forms renal calculi composed of mainly calcium oxalate (Bahuguna et al., 2009). The mechanism for this process may be due to an increase
in the urinary concentration of oxalates. The increased urinary calcium is a factor favoring the nucleation and precipitation of calcium oxalate from urine and subsequently crystal growth (Lemann et al., 1991). According to recent reports, the pomegranate is rich in polyphenols, anthocyanins (Vidhan et al., 2010) and many alkaloids which can cause smooth muscle relaxation specifically to the urinary and billiary tract which could facilitate the expulsion of stones from both kidneys (Calixto et al., 1984). Pomegranate is a polyphenol rich fruit, which showed potential as an anti-inflammatory agent in several experimental models (Lee et al., 2010). Treatment with PGCE and PGME decreases the calcium, phosphates and oxalates levels (Gr. IV–IX, Table 1). This may be due to anti-inflammatory effect of these compounds present in PG fruit which reduces the inflammation caused by ethylene glycol induced urolithiasis. The PGCE shows much significant decrease as compared to PGME. This suggests that the active compound in PG may be lipophilic in nature which decreases the oxalate concentration. These findings were supported by histopathological studies with paraffin kidney sections. In urolithiasis, nonprotein nitrogenous (NPN) substances such as creatinine, urea and uric acid accumulate in the blood. In this study, we find that the concentration of NPN substances viz. creatinine, urea and uric acid increases in the serum of ethylene glycol treated control rats (Gr. II, Table 2). This suggests that the EG causes renal tubular damage and decreases GFR. The groups (IV–VI, Table 2) suggests that PGCE treated rats brings significant (vs. Gr. II) decrease in the urea and uric acid to the normal limit at the dose of 200 mg/kg and it further significantly decreases to below normal (Gr.-I) levels at the doses of 400 mg/kg. However, creatinine concentration decreases significantly (vs. Gr.-II), but it does not become normal. In PGME treated rats (Gr.-VII–IX, Table 2), the urea concentration becomes normal at the doses of 400 mg/kg, where as uric acid was found below normal and creatinine decreased significantly. This may be due to the muscular damage caused by oxidative stress in experimental rats. These findings suggest that the kidney function were improved in PG treated rats. The enhanced GFR may be attributed due PG’s potent antioxidant and anti-lipid per-oxidative property, as observed by (Bagri et al., 2009). Many workers have reported that Punica granatum fruit is rich in diverse classes of potentially antioxidant compounds such as -carotenes, vitamin C, vitamin E (Turk et al., 2008). Human, animal and cellular studies have shown that exposure to high levels of oxalate and CaOx crystals produce cellular injury mediated by membrane lipid per-oxidation through intracellular ROS (Reactive oxygen species) generation. The lithogenic effect caused by ethylene glycol must be mainly attributed to the oxidative damage caused by the high level of oxalate generated by EG. Therefore, a reduction in renal oxidative stress could be another effective therapeutic approach in the treatment of urolithiasis.
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Table 2 Effect of PGCE and PGME on serum creatinine, urea and uric acid and renal oxalate levels in ethylene glycol induced urolithiasis in rats. SI no
Groups
Creatinine (mg/dl)
1. 2. 3. 4. 5. 6. 7. 8. 9.
Normal Control Standard PGCE 100 mg/kg PGCE 200 mg/kg PGCE 400 mg/kg PGME 100 mg/kg PGME 200 mg/kg PGME 400 mg/kg
0.698 10.54 1.572 7.761 4.676 1.549 3.319 2.961 1.561
± ± ± ± ± ± ± ± ±
0.081a 0.324b *** 0.103c *** 0.446c *** 0.489c *** 0.198c *** 0.351c *** 0.319c *** 0.219c ***
Urea (mg/dl) 60.51 114.8 48.86 96.48 62.87 59.05 88.00 73.80 53.86
± ± ± ± ± ± ± ± ±
6.240a 1.049 b *** 2.049c *** 1.247c *** 1.934c *** 2.915c *** 1.130c *** 2.102c *** 1.853c ***
Uric acid (mg/dl) 1.989 3.639 2.696 2.628 1.426 1.108 2.608 2.657 1.397
± ± ± ± ± ± ± ± ±
0.141a 0.231b *** 0.230c * 0.296c ** 0.227c *** 0.154c *** 0.233c ** 0.076c ** 0.168c ***
Renal oxalate 9.345 18.470 9.220 9.842 8.595 7.583 11.230 10.450 8.648
± ± ± ± ± ± ± ± ±
0.284a 0.499b *** 0.436c *** 0.377c *** 0.853c *** 0.794c *** 0.483c *** 0.831c *** 0.717c ***
All value are mean ± SEM (n = 6) One way analysis of variance test (ANOVA) followed by Dunnette’s multiple comparison test. PGCE; Punica granatum chloroform extract; PGME; Punica granatum Methanol extract; ‘a’ is compared with ‘b’ and ‘b’ is compared with ‘c’. * P < 0.05 statistically significant. ** P < 0.01 statistically significant. *** P < 0.001 statistically significant.
Figs. 1–9. Fig. 1. Normal: Arrow marks indicate normal glomerular structure and Renal tubules. Fig. 2. Control: Arrow marks indicate Oxalate renal stone and tubular dilation and renal tubular damage. Fig. 3. Cystone: Arrow mark indicate little tubular dilation and normal glomeruli. Fig. 4. PGCE 100 mg/kg, arrow marks indicate Interstitial and glomerular atrophy and hemorrhage, when compare to control groups. Fig. 5. PGCE 200 mg/kg, arrow marks indicate tubular dilation and Bowmen’s capsule space dilated and partial atrophy of glomeruli. Fig. 6. PGCE 400 mg/kg, arrow marks indicate little tubular dilation and normal glomeruli. Fig. 7. PGME 100 mg/kg, arrow mark indicate Bowmen’s capsule space with tubular dilation. Fig. 8. PGME 200 mg/kg, arrow mark indicate partial atrophy of glomeruli little tubular dilation and normal glomeruli. Fig. 9. PGME 400 mg/kg, arrow mark indicate little tubular dilation and normal glomeruli.
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Recently, it was shown that the effects of Punica granatum juice on hyperoxaluria-induced in the rats kidney, shown that the inhibitory effects on renal tubular cell injury and oxidative stress caused by oxalate crystals by reducing ROS, iNOS (Inducible nitric oxide synthase) and nuclear factor-kappaB expression (Tugcu et al., 2008). The decreased concentration of creatinine, urea and uric acid in serum and calcium, phosphate and oxalate in urine and no oxalates deposited in renal tissue, in PGCE and PGME treated rats were attributed to rich antioxidants present in these extracts. Vitamin E may prevent calcium oxalate crystal deposition in the kidney by preventing hyperoxaluria-induced per-oxidative damage to the renal tubular membrane surface, which in turned can prevent calcium oxalate crystal attachment and subsequent development of the kidney stones (Touhami et al., 2007). 6. Conclusion The administration of PGCE and PGME with EG induced urolithiatic rats resulted in removal of deposition of CaOx crystals into kidneys, improving renal histology and GFR. The observed action of the PG may be due to the prevention of urinary supersaturation, inhibition of mineralization of stone-forming constituents, normalization of the cellular function by neutralizing the effect of ROS, which could have caused oxidative stress in renal tubules. The study suggest that antioxidants, polyphenols and alkaloid of PG is therapeutically effective for the treatment of calcium oxalate stones, exhibiting effects through a combination of antioxidant and anti-inflammatory action, which could be responsible for its antilithiatic activity. Further, control studies are necessary to know the mechanism of action of PG in treatment of urolithiasis. Acknowledgements The authors are extremely thankful to Dr. V.D. Domble, Prof. & HOD of Pathology department, S.N Medical College, Bagalkot for his valuable comments on histopathological study and M/S Himalaya drug company for providing free drug sample of Cystone for this study. References Alessandra, C.P., Elvino, J.G.B., 2003. Dietary calcium intake among patients with urinary calculi. Nutritional Research 23, 1651–1660. Asish, K.D., Subhash, C.M., Sanjay, K.B., Sanghamitra, S., Das, J., Saha, B.P., Pal, M., 1999. Studies on antidiarrhoeal activity of Punica granatum seed extract in rats. Journal of Ethnopharmacology 68, 205–208. Bagri, P., Ali, M., Aeri, V., Bhowmik, M., Sultana, S., 2009. Antidiabetic effect of Punica granatum flowers: effect on hyperlipidemia, pancreatic cell lipid peroxidation and antioxidant enzymes in experimental diabetes. Food and Chemical Toxicology 47, 50–54. Bahuguna, Y., Rawat, M.S.M., Juyal, V., Gupta, V., 2009. Antilithiatic effect of flower of Jasminum Auriculatun Vahl. International Journal of Green Pharmacy 3, 155–158. Ballahb, B., Chaurasia, O.P., Ahmed, Z., Singh, S.B., 2008. Traditional medicinal plants of cold desert Ladakh-used against kidney and urinary disorders. Journal of Ethnopharmacology 118, 331–339.
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