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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

PROTECTIVE EFFECT OF SPIRULINA ON ETHYLENE GLYCOL INDUCED UROLITHIASIS IN RATS J.Sunitha1, S.Asha1*, G.Taju2

1Department of Biochemistry, D.K.M.College for Women, (Autonomous), Sainathapuram,Vellore, Tamil nadu, India 2Aquaculture Biotechnology Division, C.Abdul Hakeem College, Melvisharam, Vellore, Tamil nadu, India

Article Received on: 11/02/12 Revised on: 20/03/12 Approved for publication: 08/04/12

*Email:asha.sivaji@gmail.com

ABSTRACT The aim of our present study is to assess the effects of spirulina as a preventive agent in experimentally induced urolithiasis model in rats. Rats were induced to produce kidney stone by providing drinking water containing 0.75% ethylene glycol (v/v) (EG) for 6 weeks. In additiion to this, spirulina extract 20mg/kg, were administered along with ethylene glycol for 6 weeks. On 24 hours after 6 weeks, the blood was collected from individual rats and used for the estimation of sodium, potassium, chloride, calcium, phosphorus, urea and creatinine. The ethylene glycol feeding resulted in an increased level of serum sodium, potassium, chloride, calcium, urea and creatinine, with a decreased level of phosphorus, compared to normal rats. All these conditions were reversed on spirulina treatment. Histopathological analysis also showed that rats treated with ethylene glycol had large deposits of calcium oxalate crystals, and that deposits were reduced in rats treated with spirulina. These data suggest that spirulina has a protective activity against urolithiasis. KEYWORDS: Urolithiatic activity, Spirulina, Ethylene glycol, Sodium, Potassium ,Chloride, Calcium. INTRODUCTION Urinary stone disease is a common disorder found to be increased in western societies for the last five decades. Kidney stone formation is a complex process and it results as a cascade of events, including crystal nucleation, growth and aggregation, crystal retention within the renal tubules1. Usually kidney stones are yellow or brown colour2with a smooth or gagged structure. Some common type of kidney stones are calcium oxalate, calcium phosphate, struvite, uric acid and cystine and among these the most common form of kidney stone in humans as well as rats is calcium stones3. Approximately 12% of the world population are estimated to be affected by urinary stone disease with a recurrence rate of 70-81% in male and 47-60% in female4-6. Extra corporeal shock-wave lithotripsy, percutaneous nephrolithotomy, ureteroscopy and nephrolithotomy are the surgical treatment used to eliminate the kidney stones. Among these treatments, extra corporeal shock wave lithotripsy (ESWL) become the standard procedure in eliminating kidney stones. However, ESWL may also cause acute renal injury, a decrease in renal function and also an increase in stone reoccurence7-9. A number of non steroidal, anti inflammantory drugs like ibuprofen or narcotics are prescribed to remove the kidney stones all over the world but causes many side effects10. Hence, therefore an alternative method was focused to cure lithosis by using medicinal plants or phytotherapy11. A number of medicinal plants have been used for the treatment of kidney stone in many parts of the world. Jasmine auriculatum valh 12, helianthus annulus linn13, hibiscus sabdarifaratlinn14, hemiaria hirsuta15 and cynodon dactylon16. Spirulina reffered as cyanobacteria or blue green algae is a gram negative bacteria with soft cell wall, consist of complex sugars and protein. It can be rod or disc shaped. Spirulina are photosynthetic with a photosynthetic pigment called phycocyanin, hence autotrophic in nature. In the present study, the objective was to investigate and to validate the antinephrolithiatic activity of spirulina extract in ethylene glycol induced urolithiasis in rats.

MATERIALS AND METHODS Chemicals All the chemicals and reagents were purchased from S.D fine chemical (mumbai, india). All these were of analytical grade. Animals Male albino rats weighing between 150-200gm were selected for antiurolithiatic activity. The animals were acclimatized to standard laboratory conditions (25±20C) and maintained under controlled conditions of 12 hours day and 12 hours night cycle. All the animals were fed with a standard pellets and allowed free access to water. Experimental protocol Twenty four animals were randomly divided into four groups containing six animals per group. Each group underwent a different treatment methods for 6 weeks. Group I: Normal rats feed with normal feed and water. Group II: Normal rats received 0.75% ethylene glycol (EG) in drinking water alone for first 3 Weeks, followed by drinking water for next 3 weeks. Group III: Normal rats received 0.75% ethylene glycol (EG) in drinking water for 3 weeks and Spirulina solution (20 mg/kg of body weight /day) for next 3 weeks. Group IV: Normal rats treated as group1 for first 3 weeks and feed with spirulina solution (20 mg/kg of body weight /day) for next 3 weeks. Spirulina preparation The air dried spirulina was mixed under cooling conditions with sterile distilled water and used. Biochemical analysis All the treatments were done once daily by orally for 6 weeks. After 6 weeks, all the animals were sacrificed under anesthesia and blood samples were collected. The collected blood samples are allowed to clot under room temperature. The serum was seperated by centrifugation at 1500rpm for 15 mins and used for the estimation of serum urea, creatinine, phosphorus, calcium, potassium, chloride and sodium. Estimation of urea by diacetyl monoxime method17, Estimation of creatinine by Jaffe’s method18, inorganic phosphorus estimated by Fiske and Subbarow method 192519, amount of calcium estimated by the method of Clark in

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192520, Determination of potassium by folin-coicalteau phenol reagent method21, chloride is determined according to the method of R.G.Schoenfeld and C.J.Jewellen22, the amount of sodium in serum was determined by E.C.Butterworth in 1950.23 Histopathological analysis After sacrifice, the abdomen was cut open to remove the kidney from each animal. Each kidney was fixed in 10% formalin solution, dehydrated, embedded in a paraffin wax, and serially sectioned using a microtome. The sections of 5µm were stained using hematoxylin-eosin (H&E) staining24. The photographs were taken using a light microscope with a camera attachment. Statistical analysis All the statistical comparison were made by means of one way analysis of variance (ANOVA) between the groups and followed by multiple comparison test. The p values of < 0.05 were considered to be significant. The results are expressed as mean ± standard deviation (S.D) for 6 rats in each group. RESULTS In the present study, chronic administration of 0.75% (v/v) ethylene glycol aqueous solution to male albino rats resulted in an increased concentration of sodium, potassium, chloride, and calcium in serum of calculi induced rats (Group II). The sodium, potassium, chloride and calcium concentration were significantly increased in calculi-induced animals (Table 1) when compared to normal. However, treatment with aqueous extract of spirulina significantly lowered the elevated levels of sodium, potassium, calcium and chloride in Group III as compared to normal animals (Table 1, Figure 1). The serum phosphorus level gets lowered in calculi induced animals (Table 1, Group II) as compared to the normal animals. The results were comparatively increased in spirulina treated rats (Group III) compared to the normal (Table 1, Figure 1). Although, the data also reveals that the group (Group IV) treated with spirulina only shows no significant changes. Similarly, the serum creatinine and urea were increased in urolithiatic animal (Group II) compared to normal (Group I) showing renal stones. The treatment with spirulina significantly (p<0.05 / p< 0.001 vs Group I) decreased the serum urea and creatinine levels. These results indicates that spirulina treatment shows some improvement in renal function (Group III) compared to urolithiatic control (Group II). (Table 2, Figure 2 ,3) Histopathological analysis revealed a normal architecture in normal and spirulina only treated rats Group I and Group IV). On the other hand, many calcium oxalate deposits and dilation of the proximal tubules were observed in the urolithiatic rats (Group II). The number of calcium oxalate deposits and dilation of proximal convoluted tubules were less in spirulina treated rats than the Group II rats. (Figure 4a, b, c, d ). DISCUSSION In the present study, the chemical ethylene glycol in the concentration of 0.75% is used to induce urolithiasis in rats25-

27. The male rats are selected to induce urolithiasis because the urinary system of male rats resembles as like that of the human28-29 . Also earlier studies have shown that the amount of stone deposition in female rats was significantly less30 . In this study, the ethylene glycol is used to induce the urolithiasis in rats. Ethylene glycol is non-toxic and gets eliminated by the kidney. Ethylene glycol gets metabolized into glycolic acid, glycoxalic acid and oxalic acid. The formed oxalic acid are largely excreted in the urine as

calcium oxalate and gets deposited in the renal tubule. Due to deposition of calcium oxalate, there is a obstruction in the urine flow in urinary system. Because of this reason, the glomerular filtration rate (GFR) also decreases. Due to this, the wasteproducts, particularly nitrogeneous substances such as urea, creatinine and uric acid gets accumulated in blood. 31 It has been already reported that the kidneys are the principle target organ for ethylene toxicity and hence administration of ethylene glycol for 3 weeks resulted in a increased calcium, potassium, sodium and chloride and deposition of crystals in kidney32-35. In addition, the recent study indicated that the significant decrease in phosphorus level in serum promotes the crystal formation in kidney as calcium oxalate. (Group II, Table 1). Treatment with spirulina decreases the calcium, potassium, sodium and chloride levels (Group IV, Table 1). But the level of phosphorus was increased by the administration of the extract36,37,35. In urolithiasis, non protein nitrogenous (NPN) substances such as creatinine and urea gets accumulated in the blood38

and hence the increased concentration in blood of ethylene glycol induced rats (Group II, Table 2). This proves that the ethylene glycol causes renal tubular damage and decreases the glomular filteration rate. The group III suggests that spirulina treated rats brings a significant decrease in the creatinine and urea level near to the normal, but, it does not become normal. These results hastens that spirulina dissolves the preformed stones and prevents the formation of new stones in urinary system in greater extent. In conclusion, the presented data indicate that administration of ethylene glycol induced lithiasis and also the spirulina extract also seems to have greater preventive effect on induced kidney stones. ACKNOWLEDGEMENT The author is thankful to Miss S.Asha, Assistant professor, for her guidance ,friend V.Sankari, B.Deevika and all friends for providing the necessary help to carry out this research work. REFERENCES 1. Coe FI, Parks JH. Pathophysiology of kidney stones and stratergies for

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3. Christina AJM, Najumadeen NAH, Kumar SV, Manikandan N, Tobin GC, Venkataraman S, Murugesh N. Antilithiatic effect of Melia azedarach on ethylene glycol induced nephrolithiasis in rats. Pharmaceutical Biology 2006; 44: 480-485.

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TABLE 1 REPRESENTS THE EFFECT OF SPIRULINA ON THE LEVEL OF SERUM SODIUM, POTASSIUM, CHLORIDE, CALCIUM AND

PHOSPHORUS IN UROLITHIASIS INDUCED RATS Parameters Group I Group II Group III Group IV

Sodium(mEq/L) 148.72 ± 4.32

160.34 ± 1.94*a 157.55 ± 0.52 152.08 ± 3.37

Potassium(mEq/L) 6.07 ± 0.24 11.42 ± 0.89 7.73 ± 0.56**a 6.00 ± 0.43

Chloride(mEq/L) 102.42 ± 2.30 109.54 ± 0.78*a 107.91 ± 0.60 101.89 ± 2.63

Calcium(mg/dL)

10.49 ± 0.63 15.50 ± 0.43 11.93 ± 0.35***a 10.11 ± 0.57

Phosphorus(mg/dL) 6.10 ± 0.33 2.67 ± 0.31 8.61 ± 0.96*a 7.68 ± 0.96

Results are expressed as mean ± S.D (n =6 animals / group ), analysed by one way analysis of variance test (ANOVA) followed by Bonforrni multiple comparison test.

*p < 0.001 statistically significant, **p < 0.01 statistically significant, ***p < 0.05 statistically significant. ‘a’ comparison are made with Group I.

TABLE 2 : EFFECT OF SPIRULINA ON SERUM CREATININE AND UREA LEVELS IN ETHYLENE GLYCOL INDUCED UROLITHIASIS IN

RATS Parameters Group I Group II Group III Group IV

Creatinine

0.42 ± 0.78 1.21 ± 0.23 0.70± 0.04**a,*b 0.50±0.05

Urea 19.86 ± 0.62 26.39± 0.84 22.95 ±1.03*a,* b 19.84±0.85* c

All values are mean ± S.D (n=6 animals / group ), analysed by one way analysis of variance test (ANOVA) followed by Bonforrni multiple comparison test. Creatinine and urea were expressed in mg/dl.

*p < 0.001 statistically significant, ***p < 0.05 statistically significant ‘a’ comparison are made with Group I. ‘b’ comparison are made with Group II. ‘c’ comparison are made with Group III.

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Figure 1 the level of sodium, potassium, chloride, calcium and phosphorus in serum of normal, ethylene glycol induced , pretreated (spirulina and

ethylene glycol )and spirulina only of 4 experimental rats

Figure 2 the level of serum creatinine in four experimental group animals – normal, ethylene glycol induced, spirulina and ethylene glycol treated, and spirulina only rats

Figure 3 depicts the level of serum urea in normal, ethylene glycol induced, spirulina and ethylene glycol treated , and spirulina only

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Figure 4a – Normal kidney Figure 4b – Ethylene glycol induced

Figure 4c – ethylene glycol + spirulina Figure -4d Spirulina only

treated Figure 4a,b,c,d represents the histopathology of normal, ethylene glycol induced, spirulina only and pretreated

(spirulina and ethylene glycol ) rats.

Source of support: Nil, Conflict of interest: None Declared