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Vol. 15, No. 3, 2009 159-166 African Journal of Urology 1110-5704 Original Article Evaluation of Pyracantha crenulata Roem for Antiurolithogenic Activity in Albino Rats Y.M. Bahuguna1, M.S.M. Rawat2, V. Juyal3 and K. Gusain1 1 Division of Pharmaceutical Sciences, Shri Guru Ram Rai Institute of Technology and Sciences, Patel Nagar, Dehradun-248 001, 2Department of Chemistry, H.N.B. Garhwal University, Srinagar, Pauri Garhwal, 3 Department of Pharmacy, Kumaun University, Bhimtal Campus, Bhimtal, Nainital, Uttarakhand, India ABSTRACT Objective: The aim of the present study was to investigate the effect of juice extract and alcohol extract of fruit of Pyracantha crenulata (D.Don) Roem (Rosaceae) against ethylene glycolinduced urolithiasis in male albino rats. Patients and Methods: Lithiasis was induced in rats by administering 0.75% ethylene glycol in drinking water for 28 days and was manifested by hyperoxaluria as well as increased renal excretion of calcium, phosphate and a low urinary magnesium content. Curative and preventive treatment was then tried by supplementation with juice and alcohol extracts (250 mg/kg b.w., p.o.) of P. crenulata fruit. Results: The increased deposition of stone forming constituents in the kidneys of calculogenic rats was significantly lowered by curative and preventive treatment using juice and alcohol extracts (250 mg/kg b.w., p.o.) of P. crenulata fruit which showed a regulatory action on endogenous oxalate synthesis. Conclusion: From this study, we conclude that both prophylactic and therapeutic treatment with juice and alcohol extract of fruit of P. crenulata may reduce precipitation of calcium oxalate, with improvement of kidney function as well as cytoprotective effect Keywords : Ethylene glycol, hyperoxaluria, nephrolithiasis, Pyracantha crenulata Corresponding Author: Dr. Yogendr M. Bahuguna, Division of Pharmaceutical Sciences, SGRR Institute of Technology and Sciences, Patel Nagar, Dehradun-248 001, Uttarakhand, India, E-mail: yogendr.bahuguna@gmail.com Article Info: Date received : 14/4/2009 Date accepted (after revision): 14/11/2009 INTRODUCTION Despite technological and conceptual developments in the practice of medicine, the formation and growth of renal calculi continue to afflict mankind. The incidence of kidney stones has increased in western societies in the last five decades, in association with economic development. Most calculi in the urinary system arise from a common compo- nent of urine, calcium oxalate (CaOx), representing up to 80% of all analyzed stones1. A large number of plant drugs have been used in India since ancient times which claim efficient cure of urinary stones2. Amongst the medicinal plants used in the treatment of urolithiasis are ‘patharphor’ (Didymocarpus 159 ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA PATIENTS AND METHODS Fresh Pyracantha crenulata fruit were collected from local areas of Dehradun, Uttarakhand, India during May 2008 and authenticated at the Botanical Survey of India (BSI), Dehradun, India. A voucher specimen of the plant was deposited in the Botanical Survey of India herbarium under the number BSD 112215 for further reference. The fresh and semi-ripe fruit were cut into small pieces and fed through a juicer. The juice was filtered and vacuum dried to obtain P. crenulata fruit juice extract (PCJE, yield 17% w/w). In addition, the fruit were sliced using a home slicer, then shade-dried, pulverized and passed through a 20-mesh sieve. The dried, coarsely powdered plant material was extracted with 70% (v/v) alcohol by the hot continuous extraction method using a soxhlet apparatus at a temperature of 6070ºC. The solvent was evaporated under vacuum yielding a semisolid mass (PCAE, yield 22% w/w with respect to the dried powder). Both extracts were stored in tight containers in a desiccator for further use16. Fig. 1: Fruits and leaves of P. crenulata pedicellata), several Bergenia species, three species of Tribulus (T. systoides, T. terrestris and T. alatus), ‘manjit’ (Rubia cordifolia and Rubia tinctorum), ‘varuna’ (Crataeva nurvala) and ‘imli’ (Tamarindus indica)3, Costus spiralis4, Raphanus sativus5, Moringa oleifera6, Crataeva adansonii7, Melia azedarach8 and Eleusine coracana9. “The plant Pyracantha crenulata Roem, syn. Crataegus crenulata Roxb (Rosaceae), locally known as Ghingaaru, is found in the Himalayas from Sutlaj to Bhutan, at altitudes of 800-2500 m. In Ayurvedic medicine it is reported to be useful in the treatment of a number of ailments, including hepatic, stomach and skin diseases due to its diuretic, depurative, tonic, antirheumatic, cardiotonic, hypoglycemic, hypotensive, anti-inflammatory and lithontripic properties10-14. However, there are no records of systematic pharmacological studies that support its antiurolithogenic effect. Due to the wide distribution in Uttarakhand, India, and in continuation of our research work on P. crenulata15, the present investigation was carried out to determinate the antiurolithogenic property of the alcohol and juice extracts of the fruit of P. crenulata using an ethylene glycol induced hyperoxaluria model in albino rats and to confirm the traditional medicinal use of the plant. For acute toxicity studies, albino rats of either sex weighing 150-200 g were selected, and healthy adult male albino rats weighing 150-200 g were selected for assessing the antiurolithogenic activity. The animals were acclimatized to standard laboratory conditions (temperature: 25 ± 2ºC) and maintained on a 12-h light: 12-h dark cycle. They were housed in polypropylene cages and provided with regular rat chow (Lipton India Ltd., Mumbai, India) and drinking water ad libitum. The animal care and experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC). The acute oral toxicity study17 was carried out as per the guidelines set by the Organization for Economic Co-Operation and Development (OECD) received from the Committee for the Purpose of Control and Supervision of Experiments on Animals 160 ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA Table 1: Urinary excretion of stone forming constituents in control and experimental animals Urine parameters (mg/dl) Groups Dose (mg/kg) Oxalate Calcium Phosphate Normal (N) Vehicle 0.28 ± 0.01 1.18 ± 0.03 2.96 ± 0.04 Calculi induced (PR) Vehicle 2.61 ± 0.17* 3.85 ± 0.18* 6.82 ± 0.13* Cystone treated (CR) 750 0.46 ± 0.10*** 1.34 ± 0.10*** 3.36 ± 0.03*** Alcohol extract (CR I) 250 0.86 ± 0.01*** 2.03 ± 0.24*** 3.40 ± 0.26*** Juice extract (CR II) 250 2.25 ± 0.24** 3.52 ± 0.31** 5.98 ± 0.28** Alcohol extract (PR I) 250 1.28 ± 0.13*** 2.27 ± 0.13*** 4.52 ± 0.22*** Juice extract (PR II) 250 2.36 ± 0.16** 3.41 ± 0.23** 6.48 ± 0.32** Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR (*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01) (CPCSEA). One tenth of the median lethal dose (LD50) was taken as an effective dose18. From the acute toxicity study, the LD50 cut-off dose was found to be 2500 mg/kg body weight for both extracts. Hence, the therapeutic dose was taken as 250 mg/kg body weight for both extracts. From day 1 - 28, group PR I received alcohol extract (250 mg/kg body weight) and group PR II juice extract (250 mg/kg body weight). Group PR did not receive any extract. From day 15 – 28, group CR received a standard antiurolithogenic drug (cystone; 750 mg/kg body weight20-23), while groups CR I and CR II received alcohol extract (250 mg/ kg body weight) and juice extract (250 mg/kg body weight), respectively. An ethylene glycol induced hyperoxaluria model19 was used to assess the antiurolithogenic activity in albino rats. The animals were divided into seven groups containing six animals each. • Group N served as a normal control group and received regular rat food and drinking water ad libitum. • Groups PR, PR I and PR II for the preventive regimen • Groups CR, CR I and CR II for the curative regimen All extracts were given orally once daily. All animals were kept in individual metabolic cages, and 24-h urine samples were collected on day 28. The animals had free access to drinking water during the urine collection period. A drop of concentrated hydrochloric acid was added to the urine before it was stored at 4ºC. The urine was analyzed for calcium24, phosphate25 and oxalate26. Starting from day 1, ethylene glycol (0.75% v/v) in the drinking water was given to all groups except the control group for the induction of renal calculi. After the experimental period, blood was collected from the retro-orbital area under 161 ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA Table 2: Kidney retention of stone forming constituents in control and experimental animals Kidney parameters (mg/g) Groups Dose (mg/kg) Oxalate Calcium Phosphate Normal (N) Vehicle 1.36 ± 0.02 3.02 ± 0.03 2.58 ± 0.04 Calculi induced (PR) Vehicle 4.85 ± 0.11* 4.94 ± 0.21* 3.88 ± 0.11* Cystone treated (CR) 750 2.15 ± 0.03*** 1.82 ± 0.27*** 1.62 ± 0.03*** Alcohol extract (CR I) 250 2.18 ± 0.03*** 2.82 ± 0.21*** 1.80 ± 0.06*** Juice extract (CR II) 250 3.42 ± 0.06** 4.26 ± 0.34** 3.47 ± 0.16** Alcohol extract (PR I) 250 4.28 ± 0.36*** 3.46 ± 0.22*** 3.02 ± 0.07*** Juice extract (PR II) 250 4.11 ± 0.23** 4.49 ± 0.32** 3.50 ± 0.16** Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR (*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01) RESULTS anesthetic conditions, and the animals were sacrificed by cervical decapitation. The serum was separated by centrifugation at 10,000 rpm for 10 min and analyzed for creatinine, urea nitrogen27 and uric acid28. In the present study, chronic administration of 0.75% (v/v) ethylene glycol aqueous solution to male albino rats resulted in hyperoxaluria. Oxalate, calcium and phosphate excretion were grossly increased in calculi-induced animals (Table 1). However, supplementation with juice and alcohol extracts of P. crenulata fruit significantly (p<0.001) lowered the elevated levels of oxalate, calcium and phosphate in urine and kidney in the animals of groups CR I and CR II (curative regimen) and those of groups PR I and PR II (preventive regimen) as compared to group PR (calculi-induced) (Table 1). The abdomen was cut open to remove both kidneys, which were dissected from extraneous tissue and preserved in 10% neutral formalin. They were then dried at 80ºC in a hot-air oven. A sample of 100 mg of the dried kidney was boiled in 10 ml of 1N hydrochloric acid for 30 min and homogenized. The homogenate was centrifuged at 2,000 rpm for 10 min and the supernatant was separated29. The calcium24, phosphate25 and oxalate26 contents in the kidney homogenate were determined. The deposition of crystalline components in the renal tissues, namely oxalate, phosphate and calcium, was increased in the stone-forming rats (Table 2). Treatment with juice and alcohol extracts of P. crenulata fruit significantly (p<0.001) reduced the renal content of these stone-forming constituents in both regimens (Table 2, CR I, CR II, PR I, PR II). The results were expressed as mean ± SD. Differences among the data were statistically analyzed using one-way ANOVA followed by Student Newman Keul’s test (GraphPad Prism software for Windows, Version 5.01.2007). Statistical significance was set accordingly30. 162 ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA Table 3: Serum parameters in control and experimental animals Serum parameters (mg/dl) Groups Dose (mg/kg) BUN Creatinine Uric acid Normal (N) Vehicle 38.21 ± 0.16 0.56 ± 0.03 1.26 ± 0.04 Calculi induced (PR) Vehicle 52.67 ± 0.46* 0.98 ± 0.08* 3.48 ± 0.11* Cystone treated (CR) 750 42.60 ± 0.38*** 0.58 ± 0.01*** 1.88 ± 0.03*** Alcohol extract (CR I) 250 40.31 ± 0.22*** 0.60 ± 0.03*** 2.06 ± 0.04*** Juice extract (CR II) 250 52.90 ± 0.42** 0.78 ± 0.07** 3.18 ± 0.08** Alcohol extract (PR I) 250 46.23 ± 0.23*** 0.61 ± 0.06*** 2.46 ± 0.06*** Juice extract (PR II) 250 51.87 ± 0.36** 0.84 ± 0.10** 3.12 ± 0.16** Values are expressed as mean ± SEM of 6 observations; Statistical comparisons are made between Group N vs Group PR and CR (*P<0.001); Group PR vs PR I and PR II (**P<0.01); Group CR vs Group CR I and CR II (***P<0.01) Although the extent of reduction was insignificant on inter-regimen comparison (curative versus preventive regimens), the differences were significant (p<0.001) when compared with the cystone-treated animals (Table 2). amount of stone deposition in female rats was significantly less31,32. DISCUSSION Urinary supersaturation with stoneforming constituents is generally considered one of the causative factors in calculogenesis. Previous studies indicate that in response to the administration of ethylene glycol (0.75%, v/v) over a 14-day period, young male albino rats form renal calculi composed mainly of calcium oxalate18,33,34. The biochemical mechanisms of this process are related to an increase in the urinary concentration of oxalate. Stone formation in ethylene glycolfed animals is caused by hyperoxaluria, which causes increased urinary retention and excretion of oxalate33. Similar results have been obtained when rats were treated with ethylene glycol and ammonium oxalate35,36. In the present study, male rats were selected to induce urolithiasis because the urinary system of male rats resembles that of humans and earlier studies showed that the In the present study, oxalate and calcium excretion progressively increased in stoneforming animals (PR). Since it is accepted that hyperoxaluria is a more significant risk Serum uric acid and blood urea nitrogen (BUN) were remarkably increased in the stone-forming animals (Table 3), while serum creatinine was only slightly elevated in group PR indicating marked renal damage. However, P. crenulata extracts significantly (p<0.001) lowered the elevated serum levels of creatinine, uric acid and BUN in the groups of curative and preventive regimen (Table 3). 163 ANTIUROLITHOGENIC ACTIVITY OF P. CRENULATA factor in the pathogenesis of renal stones than hypercalciuria, the changes in urinary oxalate levels are relatively more important than those of calcium37,38. Increased urinary calcium is a factor favoring the nucleation and precipitation of calcium oxalate or apatite (calcium phosphate) from urine and subsequent crystal growth39. However, juice and alcohol extracts of P. crenulata fruit lowered the levels of oxalate as well as calcium excretion. In conclusion, the presented data indicate that administration of juice and alcohol extracts of P. crenulata fruit to rats with ethylene glycol-induced lithiasis reduced and prevented the growth of urinary stones, thus supporting folk information regarding the antiurolithogenic activity of the plant. The mechanism underlying this effect is still unknown, but is apparently related to increased diuresis and lowering of urinary concentrations of stone forming constituents. These effects could explain the antiurolithogenic property of P. crenulata. An increase in urinary phosphate is observed in stone-forming rats (PR). Increased urinary phosphate excretion along with oxalate stress seems to provide an environment appropriate for stone formation by forming calcium phosphate crystals, which epitaxially induces calcium oxalate deposition40. Treatment with P. crenulata fruit extract restored the phosphate level, thus reducing the risk of stone formation. Acknowledgements The authors express their thanks to Botanical Survey of India Dehradun, Uttarakhand, India for authentification of the plant material. The authors also express their gratitude to Shri Mahant Devendra Dass Ji Maharaj, Chairman, Shri Guru Ram Rai Institute of Technology and Sciences, Dehradun, Uttarakhand, India for providing the facilities necessary to carry out the research work. In urolithiasis, the glomerular filtration rate (GFR) decreases due to obstruction of urine outflow by stones in the urinary system. Due to this, the waste products, particularly nitrogenous substances such as urea, creatinine, and uric acid accumulate in the blood41. Also, increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi-producing diet42,43. 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Effect of DL alpha-lipoic acid on tissue lipid peroxidation EDITORIAL COMMENT This study is interesting and should be published, as based on the investigations and results presented by the authors Pyracantha crenulata may gain clinical importance for the inhibition of urinary calculi. However, clinical studies on patients with urinary calculi are still necessary to confirm the said effect in humans Dietmar Bach Prof. emerit. Rheinische Friedrich Wilhelm Universität Bonn, Germany 166