Evaluate Of Antiurolithitic Activity Of Ethanolic Essay

Zalavadiya Sohin K (M. Pharm, Gujarat Technological University, Gujarat, India),

RachchhaManish A (Ph.D)

JadavPinakin D (M. Pharm, Gujarat University, Gujarat, India),

Department of pharmacology,

S. J. Thakkar Pharmacy College, Kalawad road, Rajkot 360 005, Gujarat, India

*Correspondence Address:

Mr. Pinakin D. Jadav

AA-24, Oscar Residensy,

Near Shiv Park, NanavatiMarg,

150 ft. Ring Road,

Rajkot-360 007

Gujarat, India

E- mail: [email protected]

Mobile: +91-9925848046

Total number of pages: 20

Total word counts for abstract:178

ABSTRACT

Objective : Evaluate of antiurolithitic activity of ethanolic extract of CyperusrotundusLinn. Rhizome.

Materials and methods:The anti urolithiatic was activity experimentally evaluated by using poly ethylene glycol (0.75% v/v)induced rats for 28 days. The level of various urolithiatic promoters in the biological samples (urine, serum and kidney homogenate) renal function &Histopathology were used as criteria for assessing the antiurolithiatic effect of EECR. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Results: EECR at a dose of 750 mg/kg produce wistar rats were divided in 4 group(n=6) control, diseases control,standard & test. significant increase in urine volume (p<0.001). In ethylene glycol induced urolithatic model, EECR showed significant increases excretion and deposition of various urolithiatic promoters (calcium, oxalate, phosphate and uric acid) as compared to urolithiatic control (p<0.05). EECR also decreased serum concentration of urea, uric acid and creatinine as compared to urolithiatic control (p<0.01).

Conclusion: The present study showed that the antiurolithiatic activity of EECR may be mainly due to its diuretic activity. The presence of phytoconstituents like flavanoids, saponins and terpenoids in EECR might be responsible for its antiurolithatic potency of Cyperusrotundus.

ORDER A PLAGIARISM-FREE PAPER NOW

xivKey words:Cyperusrotundus,Antiurolithiatic, Ethylene glycol.

INTRODUCTION

Kidney stone is one of the oldest and wide spread disorder related excretory system known to man. Though various kinds of stone have been identified, calcium oxalate stones is the most common in humans[1].

Urinary stone is a common disorder with 70–81% rate in male and 47–60% in female[2].even though the technological development in the present medical practice ,the information and roth of renal calculi continue to effect humankind.In, 80% patient posses making of calcium oxalate stones analyzed stone3. Kidney stone formation is a complex process that results from a succession of several physicochemical events including supersaturation, nucleation, growth aggregation and retention within the renal tubules[4,5].

Kidney stone formation or urolithiasis is a complex process which is mainly occurs due to imbalance between inducer and inhibitors further in the kidneys. The recurrence of urolithiasis. even more a serious problem in patients those who have formed one stone previouslly in kidney The standard drugs used to prevent urolithiasis are not prooven to be effective , and many of them have adverse effects that compromise theirlong term use. The present day management of nephrolithiasis with open renal surgery is rarely used procedure.Evaluate Of Antiurolithitic Activity Of Ethanolic Essay. Extracorporeal Shock Wave Lithotripsy (ESWL) has now become the standard procedure for eliminating renal stones.

However, in addition to the traumatic effect of ESWL, there is retenation of persistent residue stone fragments and acute renalinjury, a decrease in renal function and an increase in stone recurrence[6]. Hence thesearch for antilithiatic drugs without side effects from natural sources has

Reqried to explore.

Cyperusrotundus Linn (Nutgrass), a grass-like plant of the family Cyperacea (Sedge family), order Cyperales or graminales[7]. The Plant is one of the most invasive weeds known, having spread out to a world-wide distribution in tropical and temperate regions. C. rotundus has been known as “the world’s worst weed” as it is known as a weed in over 90 countries and infests over 50crops worldwide.

A number of pharmacological and biological activities including antidiabetic, antidiarrhoeal, cytoprotective, antimutagenic, antioxidant, antimalarial, antipyretic and analgesic activities have been reported for this plant[8]. The rhizome part of Cyperusrotundusis common used for treatment of dysmenorrheal and menstrual irregularities.

The phytochemical investigation of Cyperusrotundusrhizome have revealed the presence of polyphenol, flavonoid, glycoside, alkaloid, saponins,sesquiterpenoids and essentialoil[9].Presence of phytoconstituents in plants like flavonoids, terpenoids, saponins, have been previously found to be responsible for diuretic activities[10].

The ethanolic extract of Cyperusrotundus Linn rhizome was found to possess Antimicrobial activity & anticonvulsant[8], hair growth activity[11], free radical scavenging activity[12],

However, the antilithiatic and diuretic activity of Cyperusrotundus Linn have not been shown in scientific research work. So, The present study is aimed to evaluate the antilithiatic activity of ethanolic extracts of Cyperusrotundus Linn rhizome. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

METHOD

Collection and identification of plant material

The rhizomes of Cyperus rotundus were purchased from Sanjivani ayurvedic store, Rajkot and the drug was identified by Department of Botany, Christ college, Rajkot.

Plant extraction

The rhizomes of Cyperus rotundus were powdered with a mechanical grinder to obtain a coarse powder. Equal quantity of powder was passed through 40 mesh sieve to get coarse powder of desired particle size. The powdered material was subjected to successive extraction with ethanol : Water (70:30% v/v) in a soxhlet apparatus at 60oC. Appearance of colourless solvent in the siphon tube was taken as the end point of extraction. The extracts were concentrated to ¾ of its original volume by distillation. The concentrated extracts were taken in a china dish and evaporated on a thermostat controlled water bath untill it forms thick paste. The extract was dried and stored in refrigerator at 4 oC in a glass bottle throughout the study. The yield was found to be 12.3 % w/w.

Drugs and chemicals

Furosemide was obtained from Aventis Pvt. Ltd (Ankleshwar, India). Cystone was obtained Himalaya . Ethylene glycol was purchased from Sulab laboratory (Baroda, India). All the chemicals used in the study were of analytical grade Sigma Aldrich (USA). All diagnostic kit were procured from Span Diagnostic Ltd (Surat ,India) . Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Instruments

Semi autoanalyser (Rayto 1904C) and cooling centrifuge (Remi C 24 Bl) were used.

Animals

Healthy, adult Wistar male rats weighing 150-200 g of equivalent age groups were procured from our instituted of S. J. Thakkar Pharmacy college of ,Rajkot. The rats were acclimatized for 3 days in cages before experiments commenced. Rats were fed with standard pellet diet and water ad libitum. They were housed in standard conditions of temperature (25±2o C), relative humidity of 45-55%, and maintained on 12-hour light: 12-hour dark cycle in the animal house of S. J. Thakkar pharmacy college, Rajkot. The experimental protocol of pharmacological study was reviewed and approved by the Institutional Animal Ethics Committee (IAEC), S. J. Thakkar pharmacy college, Rajkot(Proposal no. SJT/55-2012). All animal experiments were carried out according to the ethical guidelines suggested by the Institutional Animal Ethics Committee.

Ethyleneglycol induced urolithiasis model[13]

Ethylene glycol (EG) induced hyperoxaluria model was used to assess the antiurolithiatic activity in Wistar rats.

The ehtanolic extract of Cyperus rotundus Linn. rhizome (EECR) was suspended in 0.5% carboxymethyl cellulose (Na-CMC) for oral administration. The solution of EECR was prepared at a dose of 750 mg/kg. Cystone (750 mg/kg p.o.) in 0.5% Na-CMC was used as the standard antiurolithiatic agent.

Animals were divided into four groups containing six animals in each group. Ethylene glycol (0.75% v/v) with 1% w/v with ammonium chloride in drinking water ad libitum for 3 days in drinking water was fed to Groups II ,III, IV for induction of renal calculi till 28th day along with treatment . Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Each group underwent a different treatment protocol for 28 days as follows:

Group I (Control): received regular rat food and drinking water ad libitum.

Group II (Urolithiatic control): Treated with Na-CMC (0.5% w/v) orally at a dose of 10 ml/kg.

Group III (Standard): Treated with standard antiurolithiatic drug, Cystone orally at a dose of 750 mg/kg from 1st day till 28th day.

Group IV (Test): Treated with EECR orally at a dose of 750 mg/kg from 1st day till 28th day.

Assessment of antiurolithiatic activity

Collection and analysis of urine

All animals were kept in individual metabolic cages and urine samples of 24 hrs was collected on 28th day. Animals had free access to drinking water during the urine collection period. A drop of concentrated hydrochloric acid was added to the urine before being stored at 4°C. After urine collection, urine volume, total urinary excretion of calcium[14] ,oxalate[15], phosphate[16] and uric acid[17] were measured.

Serum analysis

After the experimental period, blood was collected from the retro-orbital under anesthetic conditions. Serum was separated by centrifugation at 10,000 rpm for 10 min and analyzed for calcium[14], phosphate[16], urea[18], uric acid[17] and creatinine[19].

Kidney homogenate analysis

The abdomen was cut open to remove both kidneys from each animal. Isolated kidneys were cleaned off extraneous tissue and preserved in 10% neutral formalin. The kidneys were dried at 80°C in a hot air oven. A sample of 100 mg of the dried kidney was boiled in 10 ml of 1 N hydrochloric acid for 30 min and homogenized. The homogenate was centrifuged at 2,000 rpm for 10 min and the supernatant was separated. The calcium[14], oxalate[15], phosphate[16] and uric acid[17] content in kidney homogenate were determined. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Histopathology

Kidney samples were weighed and fixed rapidly with 10% neutralized formalin (pH 7.4). Sections of kidney fixed in paraffin were prepared and stained with hematoxylin and eosin and observed for pathological changes. Photographs was be taken of prepared sample slides[20].

Statistical analysis

All values were expressed as Mean ± SEM (standard error of mean). The statistical analysis was done by analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Value of p < 0.05 was considered as significant. The statistical software used was Graphpad Prismversion 5.0 (GraphPadSoftware,Inc.,USA).

RESULTS

Physiological parameters

The urinary output of the control group was recorded on the 28th day, which was significantly decreased in urolithiatic control. EECR treated group showed significant increase in urinary output as compared to urolithiatic control. It was comparable to significant diuresis produced by CYSTONE, as compared to the urolithiatic control (Table 1). Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

There were significant increase in dry and wet weight of kidney in urolithiatic control as compared to the control group which was significantly prevented in CYSTONE and EECR treated groups (Table 1).

Urine analysis

The urinary excretion of various urolithiatic promoters, calcium, oxalate, phosphate and uric acid were recorded for control group. There was significant increase in urinary excretion of calcium, oxalate, phosphate and uric acid in urolithiatic control as compared to control. CYSTONE and EECR treated group showed significant decrease in urinary excretion of calcium, oxalate, phosphate and uric acid as compared to urolithiatic control (Table 2).

Serum analysis

The serum concentration of various urolithiatic promoters, calcium, phosphate and uric acid were recorded for control group. There was significant increase in serum concentration of calcium, phosphate and uric acid in urolithiatic control as compared to the control group. CYSTONE and EECR treated groups showed significant decrease in serum concentration of calcium, phosphate and uric acid as compared to urolithiatic control (Table 3).

Kidney homogenate analysis

The deposition of the urolithiatic promoters in the renal tissues, namely calcium, oxalate, phosphate and uric acid were recorded for control group. There was significant increase in the deposition of the urolithiatic promoters in the renal tissues, namely calcium, oxalate, phosphate and uric acid in urolithiatic control as compared to thecontrol group. CYSTONE and EECR treated groups showed significant decrease in the deposition of the urolithiatic promoters in the renal tissues as compared to urolithiatic control (Table 4). Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Renal function

The serum concentration of urea, uric acid and creatinine were recorded for control group. There was significant increase in serum concentration of urea, uric acid and creatinine in urolithiatic control as compared to control group indicating marked renal damage. CYSTONE and EECR treated group showed significant decrease in serum concentration of urea, uric acid and creatinine as compared to urolithiatic control (Table 5).

Histopathology of kidney

The histopathological observations of kidney of vehicle control rats showed normal structure and architectural intactness without any apparent damages (Fig 1: A). Kidney of ethylene glycol induced urolithic rats showed accumulation of calcium oxalate crystals in the tubules that led to dialation of the tubules along with interstitial inflammation (Fig 1: B). Rats co-treated with the higher dose of EECR (750 mg/kg) in showed less number of deposits of calcium and tubules than urolithic control group (Fig 1: D).and also less dilated revealed normal structured of tubules same as in Cystone treated group animal (Fig 1 : C).  Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

DISCUSSION

Formation of urinary calculi at any level of the urinary tract is a common condition. The most serious problem for this disease is the high recurrence rate. The recent treatment procedures like surgical removal, percutaneous techniques and extracorporeal shock wave lithotripsy are prohibitively costly for the common man and with these procedures recurrence is quite common and the patient has to be subjected to careful follow up for number of years. Herbal medicine is cost effective, can be affordable by all the sections of society and reduces the recurrence rate. Diuretics also reduce the risk of stone formation. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay. The disease is frequently rising in the western countries, attributed to their life style and dietary habits. Herbal drugs claim many promising remedies in urolithiasis. But scientific evaluation has been done only.

For An attempt has been made in the present study to evaluate the antiurolithiatic activity of EECR at the dose of 750 mg/kg.

Urinary supersaturation is generally considered to be one of the causative factors in calculogenesis. Imbalance between urolithiatic inhibitors and promoters in the kidneys also contribute to urolithiasis.

Rats are the most frequently used animals in models of calcium oxalate deposition in the kidneys, a process that mimics the etiology of kidney stone formation in humans[21].

In the present study, Wistar male rats were selected to induce urolithiasis because the urinary system of male rats resembles that of humans[22] and also earlier studies have shown that the amount of stone deposition in female rats was significantly less[23].

Rat models of calcium oxalate urolithiasis induced by either ethylene glycol (EG) alone or in combination with other drugs such as ammonium chloride, are often used to study the pathogenesis of kidney crystal deposition[24].

It has been reported that the kidneys are the principle target organ for ethylene glycol toxicity and administration of ethylene glycol more than four weeks resulted in insignificant urinary oxalate excretion and deposition of crystals in kidney[25], hence in our study ethylene glycol was chosen to induce urolithiasis.

Evidences in previous studies indicated that in response to 14 days period of ethylene glycol (0.75%, v/v) administration, young male albino rats form renal calculi composed mainly of calcium oxalate. The biochemical mechanisms for this process are related to an increase in the urinary concentration of oxalate. Stone formation in ethylene glycol fed animals is caused by hyperoxaluria, which causes increased renal retention and excretion of oxalate. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Weight loss observed in urolithiatic control is due to anorexia & due to disturbances in carbohydrates, proteins or fat metabolism which is affected by the ingestion of ethylene glycol. Indeed the toxic nature of oxalic acid and its nephrotoxicity was well recognized in the nineteenth century[26]. Its injurious effects are considered to be a result of the physical properties of its calcium salts (calcium oxalates) which produce at physiological pH inducing calcium oxalate nephrolithiasis. However other study indicated that free oxalate levels (above 140µM) led to increased DNA fragmentation, membrane permeability and produced a number of morphological changes including cytoplasmic vacuolization and nuclear pyknosis[27]. This injury may be exaggerated in the presence of calcium oxalate crystal. EECR produced a gain in the body weights as compared to urolithatic group ,which reveled improved.

An increase in dry and wet kidney weight were observed in urolithiatic control. Increased deposition of various urolithiatic promoters in kidney produces an increase in kidney weight.[28] However, Damage due to precipitate Calcium oxalate deposition kidney, Decrease in EECR treatment decreases kidney weight. This decreases in weight of kidney may be due toinhibation of deposition of urolithatic promoter by treatment of EECR.

It is reported that the increase in the urinary excretion volume (diuresis) facilitates the removal of small crystals and reduces the chance of these crystals to grow or aggregate which has been explained for certain herbal exhibiting antiurolithiatic activity[29]. In this study, EECR has shown significant diuretic effect at a dose of 750 mg/kg and thereby hastens the process of dissolving the preformed stones and prevention of new stone formation in urinary system.

In the present study, oxalate and calcium excretion are progressively increased in urolithiatic control. Since it is accepted that hyperoxaluria is a far more significant risk factor in the pathogenesis of renal stones than hypercalciuria[30]. The changes in urinary oxalate levels are relatively much more important than those of calcium[31]. Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.EECR lowered urinary excretion of oxalate.

Further Increased urinary calcium is a factor favoring the nucleation and precipitation of calcium oxalate or calcium phosphate from urine and subsequent crystal growth[32]. EECR lowers urinary excretion of calcium. Thus, EECR treatment may useful to reduces stone formation in kidney.

An increase in urinary excretion of phosphate was observed in urolithiatic control. 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 deposition[33]. Treatment of EECR reduces phosphate level, thus reducing the risk of stone formation.

An increase in urinary excretion of uric acid was observed in urolithiatic control. Uric acid is known to promote calcium oxalate crystal growth[34]. The predominance of uric acid crystals in calcium stones and the observation that uric acid binding proteins are capable of binding to calcium oxalate and modulate its crystallization also suggests its primary role in stone formation[35]. However, EECR treatment decreases urinary excretion of uric acid.

The ability of EECR to decrease excretion and deposition of various promoters may be due to the disintegration of mucoproteins, which are actual promoters of crystallization. A similar mechanism for antiurolithiatic agents was reported previously[36].

In urolithiasis, the glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in urinary system. Due to this, the waste products, particularly nitrogenous substances such as urea, uric acid and creatinine get accumulated in blood. In urolithiatic rats, marked renal damage was seen by the elevated serum levels of urea, uric acid and creatinine. Treatment with EECR significantly lowers serum level of urea, uric acid and creatinine. This evidens suggest that EECR treatment significationly improve renfucation in PEG induced urolithatic animal.

Phytochemical analysis of EECR indicates the presence of tannins and saponins. Saponins brings about the disintegration of mucoproteins,which are the promoters of crystallization. Tannins complex calcium thus prevent the stone formaton[37]. In the present study, the antiurolithiatic activity of EECR may be related to the disintegration of mucoproteins by saponins and the complexation of tannins with calcium.

Apparently, the antiurolithiaticeffet of EECR may be related to diuretic activity and disintegration of mucoproteins.

All these observations enabled us to confirm the antiurolithiatic potential of Cyperus rotundus rhizomes on ethylene glycol induced urolithiasis. Further more effect required to identify important phytoconstitued of Cyperus rotundus rhizomes which are produces protective effect agonist urolithasis in human.Evaluate Of Antiurolithitic Activity Of Ethanolic Essay.

Acknowledgements

We are thankful to the Principal and Management, S. J. Thakkar Pharmacy College, Rajkot, for extending laboratory competence and providing necessary amenities to carry out this work.