BIOLOGICAL EVALUATION OF INVIVO DIURETIC, AND ANTIUROLITHIATIC ACTIVITIES OF ETHANOLIC LEAF EXTRACT OF SACCHARUM OFFICINARUM

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1 Page2232 Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: BIOLOGICAL EVALUATION OF INVIVO DIURETIC, AND ANTIUROLITHIATIC ACTIVITIES OF ETHANOLIC LEAF EXTRACT OF SACCHARUM OFFICINARUM M.N.Palaksha 1 *, K.Ravishankar 2, V. Girija Sastry 3 1 Research Scholar, Department of Pharmacy, JNTUK, Kakinada. 2 Sri Sai Aditya Institute of Pharmaceutical Sciences and Research, Surampalem, E.G.Dist University College of Pharmaceutical Sciences, Visakhapatnam ARTICLE INFO Article history Received 04/06/2015 Available online 30/06/2015 Keywords Saccharum Officinarum, Diuretic, Antiurolithiatic Activity. ABSTRACT The present work was investigated to evaluate the diuretic and antiurolithiatic activities of ethanolic leaf extract of Saccharum officinarum. The ethanolic leaf extract was administered to experimental rats orally at a dose of 200mg/ kg and 400mg/kg. Furosemide (5mg/kg) was used as standard for diuretic activity. The parameters measured for diuretic activity was total urine volume; urine electrolyte concentrations such as sodium, potassium, chloride and bicarbonates. In in-vitro antiurolithiatic activity, Calcium oxalate crystallization was induced by the addition of 0.01M sodium oxalate solutions in synthetic urine. The effect of extract (100, 200 and 500μg/ml) was studied by time course measurement of absorbance at 620 nm for ten minutes by means of a spectrophotometer. Ethanolic extract showed inhibition at 120 sec (40.31, and %), and maximum inhibition of the crystallization of calcium oxalate at 600 seconds (55.24, and 72.72%). In in-vivo antiurolithiatic activity, urolithiasis was induced in male rats by administering ethylene glycol (0.75% v/v) in drinking water for 28days and the parameters such as oxalate, calcium and phosphate were estimated in urine. Serum creatinine, calcium and uric acid were also estimated. Treatment with the leaf extract of Saccharum officinarum restored all biochemical, urinary parameters. The results obtained justified the importance of the leaf extract of Saccharum officinarum as a diuretic and antiurolithiatic agent. Corresponding author M.N.Palaksha Research scholar, Department of pharmacy, JNTUK; Kakinada , palaksha.mn@gmail.com. Please cite this article in press as M.N.Palaksha et al. Biological Evaluation of Invivo Diuretic, and Antiurolithiatic Activities of Ethanolic Leaf Extract of Saccharum Officinarum. Indo American Journal of Pharm Research.2015:5(06). Copy right 2015 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

2 Page2233 INTRODUCTION The kidneys are bean-shaped organs that are present in vertebrates. They remove excess organic molecules from the blood, and removal of waste products of metabolism. Kidneys are essential to the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid base balance, and regulation of blood pressure. Diuretics are agents which augment the renal excretion of sodium and either chloride or bicarbonate primarily and water excretion secondarily. The term saluretic is some time used to describe a drug that increases the renal excretion of sodium and chloride ions [1]. Diuretics are helpful in situations like hypercalciuria, edema, acute and chronic renal failure, cirrhosis of liver, and acts as an antihypertensive agent. A number of diuretics like thiazides, furosemide, mannitol, and ethacrinic acid are used in practice[2]. Drug-induced diuresis is beneficial in many life-threatening disease conditions such as congestive nephritic syndrome, heart failure, cirrhosis, hypertension, renal failure and pregnancy toxaemia[3]. However, many of the diuretics currently used in market have been associated with a number of adverse effects includes onset of diabetes, metabolic disorders, activation of rennin angiotensin and sexual dysfunction. So it is important to consider a new alternative drug with high efficacy and fewer side effects. Urolithiasis is the formation of stones in the urinary tract that prominently cause variable degree of pain, bleeding, and further may lead to secondary infection. It is one of the third most common disorder found in humans[4]. The process of formation of kidney stones may be due to nucleation, aggregation and crystal growth phenomena[5]. Saccharum officinarum (Family-Poaceae) commonly known as sugarcane is widely cultivated throughout tropic and subtropic regions. It is a folk remedy for diarrhoea, dysentery, eyes, fever, arthritis, bedsores, boils, cancer, colds, cough, opacity, skin sores, sore throat, hiccups, inflammation, laryngitis, spleen, tumors, and wounds[6]. The plant kingdom still holds many species of plants containing medicinal value substances which have yet to be discovered and large numbers of plants are constantly being screened for their pharmacological value in addition to the already exploited plants[7]. The present work was aimed to evaluate the Saccharum officinarum leaf extract for their diuretic and antiurolithiatic activities. MATERIALS AND METHODS: Collection of plant parts: The whole plant of Saccharum officinarum was collected from the surroundings of Surampalem, East Godavari dist, Andhrapradesh. The plant was identified and authenticated by the taxonomist Mr.T.V.Raghavarao, Department of Botany SRVBSJB Maharanee College, Peddapuram, E.G.Dist. Andhra Pradesh. Preparation of Saccharum officinarum extract: The leaves were shade dried, powdered and sieved through 40mesh. The powdered leaves were extracted with Ethanol in soxhlet apparatus for 3days at 50 0 c. The obtained extract was evaporated under vacuum to evaporate the solvent completely and obtained gummy exudates, stored at low temperature in refrigerator for further studies. Diuretic Activity: Diuretic activity was determined following the Lipschitz method 8. The Institutional Animal Ethical Committee approved the experimental protocol (1269/a/10/CPCSEA). The rats [24 nos. ] were fasted for 18hrs and deprived of water prior to the experiment. A priming dose of 25ml/kg of Normal saline was given to all rats.the rats were grouped in to 4 groups [6 rats in each group]. Group I: Control group and treated with vehicle, 0.5% acacia orally. Group II: Treated with standard drug Furosemide (5mg/kg orally) dissolved in vehicle. Group III & IV: Were treated with ethanolic leaf extract of Saccharum officinarum (200mg/kg and 400mg/kg orally respectively). After drug administration the rats were placed in metabolic cages immediately, one rat per cage. The funnel and beaker were provided with cages for urine collection and a mesh to separate the faeces from the urine. The bladder was emptied by pulling the base of each rat tail[9]. The urine was collected into a beaker covered with aluminum foils to avoid evaporation. The volume of urine collected was recorded after 5hrs and urine was subjected to analysis for determination of sodium, potassium ions by Flame photometry[10] and chloride, bicarbonate by titrimetric analysis[11] after 24 hrs. Antiurolithiatic activity: Invitro-Antiurolithiatic activity: Experimental Protocol: The effect of extract on Calcium Oxalate crystallization was determined by the time course measurement of turbidity changes due to the crystallization in artificial urine on addition of 0.01Molar sodium oxalate solution. The Precipitation of calcium oxalate at 37 C and ph 6.8 has been studied by the measurement of turbidity at 620 nm using UV/Visible spectrophotometer [12]. Preparation of synthetic urine: For preparation of synthetic urine 8.5gm of sodium chloride, 3.8gm of potassium chloride, 24.5gmof urea 1.03gm of citric acid, 0.34gm of ascorbic acid, 1.18gm of potassium phosphate, 1.4gm of creatinine, 0.64gm of sodium hydroxide, 0.47gm of sodium bicarbonate and 0.28ml of sulfuric acid were added in 500ml of deionized water and stirred for 1hour. The synthetic urine was stored at -4 0 c until further use[13].

3 Page2234 Study without inhibitor: A volume of 1.0ml of artificial urine was transferred into the cell and 0.5ml of distilled water added to it and blank reading was taken. The 0.5ml of 0.01Molar sodium oxalate was added, to the previous volume and the measurement is immediately recorded for a period of ten minutes. Study with inhibitor: The extract was dissolved in distilled water filtered through membrane filter and the concentration of 100, 300 and 500µg/ml was obtained. A mixture of 1ml of artificial urine and 0.5ml of extract solution was taken in the cell. A blank reading was taken and then 0.5ml of 0.01M sodium oxalate solution was added and immediately absorbance was measured for a period of the 10 minutes with 2 min interval at 620nm[14]. The % of inhibition was calculated using the following formula: absorbance of control absorbance of test % inhibition = 100 absorbance of control In-vivo Antiurolithiatic activity: Ethylene glycol-induced hyperoxaluria method [15] was used to determine the antiurolithiatic activity in male albino rats. Rats were divided into four groups each group containing six animals. Ethylene glycol (0.75%v/v) in drinking water was given to all groups for induction of renal calculi for 28days.The extract was assessed for antiurolithiatic activity for its curative action in urolithiasis. In this experiment the extract was given from 15th day to 28th day. Group I: Normal Control group received regular rat food and drinking water. Group II (Calculi-induced): Positive control received ethylene glycol (0.75% v/v) in drinking Water for induction of renal calculi for 28 days. Group III: Received ethylene glycol and standard anti urolithiatic drug Cystone (750 mg/kg b.w) from 15th day to 28 th day[16].group IV: Received 400mg/kg of extract from 15th day to 28th day. Estimation of biochemical parameter: At the end of experiment (28th day), urine was collected and urine was subjected to analysis for determination calcium, phosphate and oxalate. Blood was collected through tail vein for determination of calcium, creatinine and uric acid in serum. Histopathological examination: At the end of experiment (29th day) all rats were anaesthetized by diethyl ether and sacrificed and the kidneys were removed for histopathological examination RESULTS Diuretic activity: The results obtained in diuretic activity were shown in table no1 & 2 Table No. 1: Diuretic effect of Saccharum officinarum leaf extracts. Sl No. Group Volume of urine(ml) after 5hrs Sodium (Na + ) Potassium (K + ) Chloride (Cl ) Bicarbonate (HCO3 ) 1 (Control) ± Furosemide 5mg/kg(standard) S. officinarum (200mg/kg) S. officinarum (400mg/kg) ± Values are expressed as Mean SEM; n=6(number of animals in each group); p< All comparisons are made with that of control. Table no.1 shows urine volume ml/5hrs and excretion of electrolytes sodium, potassium, chlorides and bicarbonate ions in urine. Ethanolic extract of leaf Saccharum officinarum shows significant diuretic effect by increasing the urine output and increased excretion of electrolytes in urine when compared to that of control.

4 Page2235 Table No.2. Saluretic, Natriuretic & Diuretic indexes of Saccharum officinarum leaf extracts. Group Saluretic Index [Na + + Cl ] Natriuretic Index [ Na + / K + ] Volume of urine in ml after 5hrs (Control) ± Diuretic Index Furosemide ± (standard) S.officinarum ± mg/kg S.officinarum ± mg/kg) Diuretic Index = {volume of urine in test group/volume of urine in control} From the results it can be observed that Table no.2 shows the saluretic, natriuretic and diuretic indices of ethanol extract of leaf Saccharum officinarum. In-vitro Anti-Urolithiatic activity: The results of In-vitro Anti-Urolithiatic activity of Saccharum officinarum leaf extracts shows dose and time dependent % inhibition were given in the below Table no. 3 and Fig: No.1. Table No. 3. In-vitro Anti-Urolithiatic activity of Saccharum officinarum extracts. Sl No. Test Concentration % inhibition at time in seconds 0 sec 120sec 240 sec 360 sec 480 sec 600 sec 1 Saccharum officinarum µg/ml 2 Saccharum officinarum µg/ml 3 Saccharum officinarum 400 µg/ml In-vitro Anti- Urolithiatic activity of Saccharum % inhibitio n Time in seconds 100µg/ml 200µg/ml 400µg/ml Fig 01: In-vitro Antiurolithiatic activity of Saccharum officinarum leaf extract. In-vivo Anti-Urolithiatic activity: Table no. 4 shows the results of In-vivo Anti-Urolithiatic activity of Saccharum officinarum leaf extracts showed dose dependent decrease in the levels of calcium, phosphate and oxalate in urine and calcium, creatinine and uric acid in serum were given in the below.

5 Page2236 S. N o. Gro up 1 Group I (Control) 2 Group II (Calculi Induced) 3 Group III (Cystone) 750mg/kg 5 GroupV S.officinaru m (400mg/kg) Table No. 4. In-vivo Anti-Urolithiatic activity of Saccharum officinarum leaf extract. Urine Calci um Phosphat e Oxalat e Serum Calcium Creatini ne Uric acid Values are expressed as Mean SEM; n=6 (number of animals in each group); p< All comparisons are made with that of control. 3.6 In calculi-induced rats, the elevated serum levels of creatinine, uric acid, and calcium indicate marked renal damage. Renal histological analysis stained with hematoxylin & eosin has given the following observations. Fig 2a: Kidneys of control rats showed normal features with prominent cortical tubules & Bowman s capsule. Fig 2b: kidneys of urolithiatic treated (0.75% EG) rats showed distorted renal tubules, marked inflammation & hemolysis.fig2c Kidneys of Cystone (750mg/kg) rats showed restored morphology as compared to. Signs of inflammation reduced, there was no hemolysis. Size of glomerulus was normal. Fig2d: Kidneys of Ethanol Ext (400mg/kg) treated rats have shown minimal signs of inflammation & hemolysis are given below Fig 02: Histopathology studies (kidney sections).

6 Page2237 DISCUSSION Previous studies have demonstrated that there are several compounds which could be responsible for the plants diuretic effects such as flavonoids, saponins [17]. The effect may be produced by stimulation or regional blood flow or initial vasodilation[18] or by producing inhibition of tubular reabsorption of water and ions[19], the result in both cases being diuresis. Ethylene glycol in liver metabolized to glycolic acid and glyoxylic acid which is further oxidized to oxalate thus promoting hyperoxaluria. The combination of uric acid, calcium, oxalate and inorganic phosphate In urine enhance crystallization [20]. Urolithiasis can be induced by Ethylene glycol to rats for 28days resulted in substantial excretion of oxalate and deposition of crystal in kidney [21]. An increased urinary calcium concentration is a factor favouring precipitation of Calcium oxalate or apatite (calcium phosphate) and subsequent crystal formation [22]. The urinary calcium excretion, decrease in serum was evident in treated urolithiatic rats. Other possible mode of action includes excessive excretion electrolytes may shows diuretic activity or decrease in the concentration of urinary salts that prevent the super saturation of the crystallizing salts. Phytochemical screening of extracts of Saccharum officinarum reported the presence of glycosides, phytosterols, saponins, tannins, flavonoids, etc[7]., Treatment with ethanol leaf extract of Saccharum officinarum leaf extract 200 and 400 mg/kg p.o significantly showed diuretic and antiurolithiatic activities on experimental rats. CONCLUSION Treatment with Saccharum officinarum leaf extract 200 and 400 mg/kg p.o significantly increase the volume of urine confirms diuretic activity and reduce the elevated calcium, phosphate,oxalate and creatinine concentrations in urine confirming the stone inhibitory effect. 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