Available online at International Journal of Toxicology and Applied Pharmacology

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1 Available online at International Journal of Toxicology and Applied Pharmacology Universal Research Publications. All rights reserved Original Article Evaluation of Coleus barbatus and Trigonella foenum graecum extracts and their combination on kidney stones in rats Javed Khan Pathan * 1, Neelam Khan 2, H.M. Sohel 1 Abstract 1. Swami Vivekanand College of Pharmacy, Indore 2. Indore Institute of Pharmacy, Indore Received 21 march 2011; accepted 28 March 2011 In the present study the effect of calcium oxalate urolithiasis urinary risk factor of extract of Coleus barbatus and Trigonella foenum graecum Roots and their combination have been studied in albino rats. By this study it is deduced that the possible effect of the extract of Coleus barbatus and Trigonella foenum graecum Roots and their combination have positive effect on the main urolithiasis risk factors Universal Research Publications. All rights reserved Key words: Urolithiasis, Ethylene glycol (EG), Coleus barbatus and Trigonella foenum graecum 1. Introduction Kidney stones, one of the most painful of the urologic disorders. Urolithiasis is the medical term used to describe stones occurring in the urinary tract. (Robbins et al 2005). Calcium containing stones, especially calcium oxalate monohydrate (whewellite),calcium oxalate dehydrate (weddellite) and basic calcium phosphate (apatite)are most commonly occurring ones to an extant of 75-90% followed by magnesium ammonium phosphate (struvite) to an extant of 10-15%,uric acid 3-10% and cystine 0.5-1%.( Dennis L. Kasper.,2002)The vast Ayurvedic literature claims a number of plants to be useful in urinary stones; still many plants need to exploited for their pharmacological actions.inspite of intensive research to establish the mechanism of stone formation, dietary management, evaluation of medicinal plants and other agents in the treatment of urinary stones. (Prasad, K.V.S.R.G., 2007) 2. Material and method 2.1. Plant material The plant materials, roots of Coleus barbatus (Laminaceae) and Trigonella foenum graecum (Fabaceae) were purchased from the market. The plant materials were identified by Dr.S.N.Mishra, Sr.scientist, Horticulture College, and Mandsaur Extraction of drugs The dried roots of Coleus barbatus and Trigonella foenum graecum were washed thoroughly to clean soil matter, before crushing in size reduction mill and reduced to moderately coarse powder. The size reduced drug was filled in soxhlet apparatus for solvent extraction. First the powdered drug was defatted with petroleum ether. Defatted drug was then dried and again filled in soxhlet apparatus for solvent extraction with solvent ethanol. Solvent was removed under reduced pressure at low temperature. Defatted drug was then dried and again filled in soxhlet apparatus for solvent extraction with solvent ethanol. (Pulok, M., 2002) 2.3. Rat treatment and urine sample collection Six groups and each group having five albino rats of either sex weighing gm. were selected. The animal were housed in cages under standard laboratory condition( in a temperature and humidity controlled room 25± 1 o C, 50% relative humidity with a 12 hr on-off life cycle) for a period of 14 days prior to the experiment. The animal received chow and tap water ad libitum. Group I was used as normal control group. Group II which was given ethylene glycol (EG) 0.75% only was used as untreated nephrolithiasis rats group. Group III was given EG 0.75% and 500mg/kg 1

2 body weight ethanolic extract of Coleus barbatus. Group IV was given EG 0.75% and ethanolic extract of Trigonella foenum graecum 200mg/kg body weight. Group V was given EG 0.75% and combination of both extract. Group VI was given EG 0.75% and standard drug (cystine) 500mg/kg body weight. EG was added in their drinking water to induce a chronic low grade hyperoxalluria and generate CaOx deposition into kidneys. Twenty four hour urine sample from each rat were collected after four weak in presence of sodium azide as antibacterial agent during four weeks to analyze some urinary components, to measure urinary volume and ph. At the end of the experimental study all animal were sacrificed after ether anesthesia. Kidneys harvested from animals were analyzed histologically after they were fixed in formalin and embedded in paraffin. (Hadizadeh, M 2007, Atmani, F., 2004, Khan, S.R., 1985) 2. Analytical procedure Urinary oxalate was estimated according to the method described by Hodgkinson et al (Hodgkinson 1970). 1 ml of urine was acidified by concentrated HNO 3 to solubelize crystals and then adjusted to ph 7 by NaOH in the presence of color indicator, the bromothymol blue. About 2 ml of saturated CaSO 4 and 14 ml of pure ethanol were added to precipitate oxalate overnight. The sample were centrifuged at 450 X g for 10 min and then filtered on filter paper. The precipitate obtained was solubilized in 10 ml of water acidified by 2 ml concentrated sulfuric acid. The samples were titrated by a solution of KMnO Sl. no. Group Table 1: Biochemical parameter of Urine Urine parameter (mg/ dl) Oxalate Calcium Creatinine 1. Normal control 3.97± ± ± Model control 11.82± ± ± Coleus barbatus (500mg/kg) 3.54±0.03** 3.81±0.10** Trigonella foenum graecum (200mg/kg) 3.68± 0.03** 3.47±0.09* 5. Combination 3.00± 0.23*** 3.04±0.02** 6. Standard (Cystone tablet 500mg/kg) 19± 0.04** 41± 0.06** 01± 0.02** 2.90± 0.04 *** 2.58±0.16*** 00±.04*** P<0.0001, the treated groups are compared with groups I and II. Values are expressed in Mean±SEM, Statistics: one way ANOVA followed by Dunnet s test, *** highly significant, ** significant and * less significant Table 2: Total urinary volume Sl.No. Groups Total urinary volume (ml) Mean± SE 1. Normal control 2.38 ± Model control 1.66 ± Coleus barbatus extract (500 mg/kg) 80 ± 0.07** Trigonella foenum graecum extract (200mg/kg) 50 ± 0.10** 5. Combination 5.00 ± 0.05 *** 6. Standard drug (500 mg/kg) 5.30 ± 0.09 *** P<0.001, the treated groups are compared with groups I and II. Values are expressed in Mean±SEM, Statistics: one way ANOVA followed by Dunnet s test, *** highly significant, ** significant and * less significant 2

3 Calcium analysis was performed by using a Merck thermo spectronic u.v.1 beam spectrophotometer equipped with a Varian hollow cathode and a deuterium background corrector. Creatinine was estimated based on principle, the production of an orange colour by the interaction of Creatinine with alkaline sodium pirate. The colour produced was compared in a colorimeter, and the Creatinine content of the urine estimated by comparison with a Creatinine solution of known concentration. (Atmani, F., 2004, Barbas, C., 2002) Table 3: Determination of urinary ph Sl.No. Groups ph of urine 1. Normal control 7.21 ± Model control 9.83 ± Coleus barbatus extract (500 mg/kg) 7.55 ± 0.12** Trigonella foenum graecum extract (200 mg/kg) 7.87 ± 0.14* 5. Combination 7.28 ± 0.11*** 6. Standard drug (500 mg/kg) 7.36 ± 0.10** P<0.001, the treated groups are compared with groups I and II. Values are expressed in Mean±SEM, Statistics: one way ANOVA followed by Dunnet s test, *** highly significant, ** significant and * less significant 2.5. Data analysis Standard evaluation was done using one-way analysis of variance (ANOVA) Statistical significance was set at P< Results are presented as mean ± standard errors (S.E.). (Hadizadeh, M 2007) 3. Results The changes in the urine parameters in the experiment animals during the study are presented (Table 1, 2, 3).The urine concentration of oxalate; calcium and Creatinine was increased significantly in animals administered with 0.75 percentage ethylene glycol. Four weeks treatment with ethanolic extract of Coleus barbatus significantly decreased urine concentration of oxalate (3.13 ±0.03), calcium (2.81±0.10) and Creatinine (16 ± 0.04) as compared to model control (oxalate ± 0.07, calcium 7.82± 0.24, Creatinine 6.97 ± 0.14) (Table 1 ).The concentration of oxalate (3.47± 0.03), calcium (3.28±0.09) and Creatinine (42 ± 0.06) of group IV treated with ethanolic extract of Trigonella foenum graecum were found to be significant as compared to model control (Group II ). The concentration of oxalate (3.03 ± 0.23), calcium (3.03±0.02) and Creatinine (09 ± 0.02) of group V treated with combination of ethanolic extract Coleus barbatus & Trigonella foenum graecum, were found to be more significant as compared to group II,group II,group IV. (Table 1). Moreover the group treated with combination of the two drugs (500 mg Coleus barbatus mg Trigonella foenum graecum) was found to be most significant from the entire group V. The percentage reductions of all parameters of urine were found more in group V and in group VI (standard). Urinary volume significantly decreased in the animals treated with the 0.75 % of ethylene glycol. Urinary volume were increased by 221(Group III), 208 (GroupIV), 240(Group V), 261(group VI) percentage compared to model control group. (Table 2) Urinary ph significantly increased in the animals treated with the 0.75 % of ethylene glycol. Urinary ph were decreased by 23 (Group III), 20 (GroupIV), 27(Group V), 25 (group VI) percentage with compared to model control group (Table 3). In histopathological observations gross examination of rats kidney from control group showed a normal cortical structure of the kidney including glomeruli and distended tubules, sclerotic glomeruli, and increased cellularity between tubules. In ethylene glycol treated group (model control group) showed cortical structure of the kidney including glomeruli and affected distended tubules, sclerotic glomeruli, and cellularity between the tubules. Group III, (500 mg ethanolic extract of Coleus barbatus) showed the recovered crypts or small sacs. Group IV, (200 mg/kg ethanolic extract of Trigonella foenum graecum) widespread focal interstitial lymphoplasmacytic infiltration with mild tubulitis, normal glomeruli and arteriolar vessels. There is sign of sclerotic glomeruli and increased cellularity between tubules. Group V (combination of both extract.) Shows minor interstitial lymphoplasmacytic infiltration in glomeruli with occasional penetration of tubular epithelium. In addition, increased cellularity between tubules is clearly visible. Group VI Standard (Cystine500 mg/kg) recovered distended tubules, sclerotic glomeruli, and increased cellularity between tubules. From the above results it was noted that the combination (ethanolic extracts of Coleus barbatus & Trigonella foenum graecum) was most significant. Discussion Urinary stone disease is mainly the result of super saturation of urine with certain urinary salts such as CaOx, the most common constituent of kidney stones. (Daudon et al 1993) Urorolithiasis is caused by several biochemical mechanisms. It is a generalized increase in the calcium content of the kidneys. (Grases, F., 1995) The major causes include those associated with an increase in the urinary levels of calcium crystals precipitation. Kidney stones usually arise because of an imbalance between the kidney s need to conserve fluid and the need to extrude waste products of low solubility. This imbalance often is precipitated by alternations in diet, fluid intake, climate and extent of physical activity. The majority of patients with calcium containing stones excretes excessive amounts of urinary calcium and often has urine that is supersaturated solution of calcium and oxalate salts ( Sarkisian M.R 2001) Many in vivo models have been developed to understand the mechanisms involved in the formation of urinary stones and to ascertain the effects of 3

4 Fig 1 Group I Normal control shows the cortical structure of the kidney including glomeruli and distended tubules, sclerotic glomeruli, and increased cellularity between tubules. Fig 4 Group IV, (ethanolic extract of Trigonella foenum graecum 200 mg/kg) widespread focal interstitial lymphoplasmacytic infiltration with mild tubulitis, normal glomeruli and arteriolar vessels. There is sign of sclerotic glomeruli and increased cellularity between tubules. Fig 2 Group II, Kidney stone Model control Shows cortical structure of the kidney including glomeruli and affected distended tubules, sclerotic glomeruli, and cellularity between the tubules. This demonstrated a thin and attenuated filtration barrier with an absence of definitive pedicels and crypts appears. Hematuria, glycosuria and renal failure are also present. Fig 5 Group V (combination of both extract.) Shows minor interstitial lymphoplasmacytic infiltration in glomeruli with occasional penetration of tubular epithelium. In addition, increased cellularity between tubules is clearly visible. The glomerular lesions are poorly characterized. Fig 3 Group III, (500 mg ethanolic extract of Bergenia ligulata) Shows extensive interstitial lymphoplasmacytic infiltration with definite tubulitis and intimal cell prominence with subintimal vacuolation in arterial vessels of kidney. This also shows the recovered crypts or small sacs. Fig 6 Group VI, Standard (Cystone 500 mg/kg): Shows the cortical structure of the kidney including glomeruli and recovered distended tubules, sclerotic glomeruli, and increased cellularity between tubul 4

5 various therapeutic agents on development and progression of the disease. Rat is the most frequent used animal to induce CaOx deposition into kidneys and mimic the etiology of the formation of stones in humans. Accordingly, it evaluated the effectiveness of medicinal plant, Coleus barbatus & Trigonella foenum graecum & their combination on rats rendered nephrolithiasis by administration of EG. In fact, the plant is widely used in India in the treatment of kidney stone formation as an antilithiasic and diuretic agent. It was noticed urinary volume increase in the rats those receiving EG and plant extracts compared to those receiving EG only. It was making a general look over data it can underline two important points. First, oxalate level remain constant in the urine of nephrolithiatic rats treated with the ethanolic extract of Coleus barbatus & Trigonella foenum graecum and their combination. While it increased in untreated rats groups. Secondly, urinary concentration of calcium and Creatinine of treated groups remained at a level comparable to control group rats. In the present study of kidney stone, after the administration of ethanolic extract of Coleus barbatus & Trigonella foenum graecum and their combination to the group of rats urine analysis shows that the occurrence of stone was decreased when compared to the kidney stone control group and combination (ethanolic extract of Coleus barbatus & Trigonella foenum graecum as effective as the standard group. Further studies are necessary to clarify the mechanism, which is still not clear so far. 5.References o Atmani, F., Slimani, Y., Mimouni, M., Aziz, M., Hecht, B., Ziyyat, A., 2004, Effect of aqueous extract from Herniaria hirsuta L. on experimentally nephrolithiasis rats. Journal of ethanopharmacology, o Barbas, C., Garcia, A., 2002, Urinary analysis of Nephrolithiasis markers, Journal of chromatography,781, o Daudon, M., Bader, C.A., Jungers, P., Urinary calculi: review of classification methods and correlations with etiology. Scanning Microscopy, 7, o Dennis L. Kasper.,2002, Principle of internal medicine, McGraw hill publication,16,1710 o Grases, F., Ramis, M., Costa-Bauza, A., March, J.G., Effect of Herniaria hirsuta and Agropyron repens on calcium oxalate urolithiasis risk in rats. Journal of Ethnopharmacology 45, o Hadizadeh, M., Khoei, A., Hadizadeh, Z., Parizady, M., 2007, Ethanolic extract of o Hodgkinson, A., (1970). Determination of oxalic acid in biological material. Clinical Chemistry 16, o Khan, S.R., 1985, Calcium oxalate urolithiasis in rat: is it a model for human stone disease. Scanning Microscopy 2, o Prasad K.V.S.R.G.,2007,Herbal Drugs in Urolithiasis, A Review,Pharmacognosy reviews,1, 175. o Pulok, M., 2002, Quality control of herbal drugs, Business horizons, New Delhi, 2,55 o Robbins, Cotran,2005,Pathologic basis of disease, 7,1012. o Sarkisian, M.R., 2001,Overview of the current animal models for human disorders, Journal of Urinology,16,201. o Satish H, Dang R, 2006, Antiurolithiatic herbal drugs-a review, Biomed, 1, o Trigonella foenum graecum on ethylene glycol induced kidney calculi, Urology journal, 4, Source of support: Nil; Conflict of interest: None declared 5