Pharmacological Evaluation of Indigenous Antiurolithiatic Formulation

The Neeri NRT-RF was evaluated for its antiurolithi a ic activity in rats. Calcium oxalate stones were induced by oral a dministration of ethylene glycol (0.75%) in male Wistar rats for 40 days, which is confirmed by the elevated level of several urina ry parameters (calcium, uric acid, magnesium in urine and urine o utput), serum parameters (calcium, creatinine, phosphorus, uric a id, magnesium, blood urea nitrogen) and kidney paramete rs (calcium, magnesium, phosphorus). Administration of Neeri NRT F for 25 days significantly decreased (to normal values) the elevated levels of serum, urine and kidney parameters and also prev ent d the deposition of crystals in tubules. Moreover, it sho wed antioxidant effect. In addition to this, NRT-RF (530.55 mg/kg) shows better results as compared to the standard drug disodium h ydrogen citrate. Furthermore, histopathology also revealed that NRT-RF treated group of rats retained no or very less crys tals deposition in the tissue, as compared to ethylene glycol treated group. © 2015 British Biomedical Bulletin. All rights reserv d Anil et al_______________________________________________________ ISSN-2347-5447 BBB[3][ 1][2015] 041-057 Introduction Urolithiasis refers to the hard crystalline minerals formed within the urinary tract . It is a common chronic disorder after the hypertension 2 and affects 10-15% population worldwide . Calcium containing stones (calcium oxalate, calcium phosphate) represent 80% of kidney stones while remaining 10% are composed of struvite, 9% of uric acid (UA); and the remaining 1% that of cystine . Kidney stone cause disabling pain, known as renal colic, which often present when stone moves from renal pelvis into ureter . Moreover stone results in bloody or foul smelling urine, nausea, vomiting like symptoms . Urolithiasis is a multicomplex process that results from supersaturation of solute in urine, nucleation, growth, aggregation and retention within the renal tubules . Supersaturation occurs when concentration of stone materials is higher in urine . There are many substances in the body known as promoters, which promote the crystal growth, while inhibitors inhibit the crystal growth. The occurrence of stone formation is also due to imbalance between the promoter and inhibitors in the kidney . The most common cause of calcium oxalate stone is Hyperoxaluria . In rat experimental model of urolithiasis ethylene glycol is taken as a metabolic precursor of oxalate production which results in hyperoxaluria . Currently no allopathic medicine is available for the treatment of kidney stone and the option is surgical procedure or high power laser, but these procedures are costly and is remained associated with high recurrence of stone formation . A number of herbs are used now a days for curing various ailments due to high benefits to risk ratio e.g. tamarind, sesame for anxiolytic & anti-depressant activity , Premna herbacea for antitumor and antimicrobial activity. Likewise there is plethora of Ayurvedic plants which have been claimed for their antiurolithiatic properties. Neeri (NRT-RF) is a polyherbal antiurolithiatic formulation which consists of number of principal herbs ( Table 1). These herbs are already mentioned for their antiurolithiatic properties and the effectiveness of Neeri has been determined by in-vivo model of ethylene glycol induced calcium oxalate stone in Wistar rats. Materials and Methods Chemicals Neeri (NRT-RF), a formulation, licensed from State Licensing Authority was procured from Aimil Pharmaceuticals India Ltd., India. Ethylene glycol (Batch no. S11S610462) was obtained from Merck Specialties private limited, India, while Citralka was procured from commercial source (Pfizer Ltd., India). Rest of the chemicals used in the experiment were of analytical grade. Animals Healthy adult male Wistar rats of age between 4-5 months, weighing 150-250g were procured from Panacea Biotec Ltd., Lalru (14050) India and housed in polypropylene cage. They were maintained under standard laboratory conditions (temperature 25±2o C with 12/12h night/dark cycle), fed with standard pellet diet (Sanjay Biological Museum, Amritsar) and water ad libitum. The experimental protocol was approved by IAEC [Protocol No. IAEC-/2012/III/0018 (PCL-M)]. The animals were acclimatized for 10 days before carrying out the experiment. The present animal experimentation work had been done as per the guidelines of CPCSEA. Phytochemical screening Preliminary phytochemical screening was carried out to detect the presence of flavonoids (Lead acetate & Sodium hydroxide Tests), glycosides (Keller Anil et al_______________________________________________________ ISSN-2347-5447 BBB[3][ 1][2015] 041-057 Killani test, Borntrager test & Legal test), steroids (Salkowaski reaction, Liberman’s reaction & Liberman’s Burchard reaction), alkaloids (Mayer’s test & Murexide test), tannins (5% FeCl 3 & Dilute HNO3 tests), carbohydrates (Fehling’s test & Benedict’s test), proteins and amino acids (Millon’s test, Xanthoprotein test & Ninhydrin test). Depending upon the plant ingredients listed on the label of the formulation. Experimental design Ethylene glycol induced hyperoxaluria method 9 was used to assess the Antiurolithiatic activity in Albino Wistar rats. Animals were divided into 5 groups with 5 animals in each group. Group I served as normal and received regular standard pellet diet and drinking water ad libitum. Ethylene glycol (0.75%) in drinking water was administered to group II to V for 40 days to induce renal calculi. Group III received oral administration of standard drug Citralka (137.7mg/kg b.w.) p.o., from 15 day till 40 day, group IV and V received 357.7 and 530.55 mg/kg oral dose of test drug (Neeri) respectively, from 15 th day till 40 day. The dosages were calculated on the basis of conversion of average of human dose to rat dose. The regimen was based on human dose of neeri. Experimental design has been shown in Table 2. Urine collection and analysis A 24 hour urine sample was collected from each group of rats using metabolic cages (Orchid scientific and innovative Pvt. Ltd. Nashik, India) on 40 th day after administration of Neeri NRT-RF polyherbal formulation. Urine output was measured. The urine was centrifuged at 3000 rpm for 10 minutes and then filtered with Whatman filter paper and then urine analysis was done for estimation of uric acid (Modified Trinder Method) , calcium (O– Cresolpthalein Complexone Method) , and magnesium (Xylidyl blue Method) 22 using diagnostic kits (ERBA) and measured by semi auto analyzer (ERBA Chem Touch, Transasia Biomedical Limited, Mumbai, India). Serum analysis The serum was obtained for biochemical analysis on 40 th day. The animals were anesthetized with diethyl ether and the blood was collected from retro orbital sinus. Serum was separated by centrifugation of blood at 3000 rpm, 4 C for 10 minutes (VCCH-3214, Semi Elektro Technik Limited, India) and analyzed for calcium, creatinine (Jaffe’s method) , uric acid magnesium, phosphorus (ammonium molybdate method) , BUN (GLDH–UREASE method) 25 using diagnostic kits ERBA and measured by semi auto analyzer. Antioxidant activity In vivo oxidative stress was assessed by measuring lipid peroxidation levels (marker of oxidative stress) in blood 26-28 collected at the end of 40 th day. Kidney harvest and measurement At the end of experiment, rats were sacrificed with deep anesthesia. Both the kidneys were removed. Isolated kidneys were cleaned off extraneous tissue and then weighed. Right kidney preserved in 10% neutral formalin and used for histopathological studies while the left kidney was dried at 80°C in hot air oven, after which the kidney was weighed and used for preparation of kidney tissue homogenate for estimation of calcium, phosphorus and magnesium contents in the kidney tissue. Anil et al_______________________________________________________ ISSN-2347-5447 BBB[3][ 1][2015] 041-057 Preparation of kidney tissue homogenate A sample of 100mg kidney was taken and homogenized by homogenizer (Popular traders, Ambala, India) and dissolved in 1N HCl overnight. The homogenate was centrifuged at 2000 rpm for 10 min and supernatant was separated. The calcium, phosphorus, magnesium content in kidney homogenate was determined by auto analyzer . Histopathological examination of kidney The kidney was weighed and washed with cold saline and preserved in10% buffered formalin. After embedding in paraffin 4 micron sections were cut in saline and stained with hematoxylin and eosin. The slides were examined under a polarizing light microscope Olympus, India. Statistical analysis The results were expressed as mean ± SEM and analyzed using Dunnett’s multiple comparison tests. Data was computed for statistical analysis using GraphPad Prism Software and p< 0.05 was considered to be statistically significant.