EVALUATION OF INVITRO ANTIUROLITHIATIC ACTIVITY OF VIDANGADI CHURNA

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Sajith et al. SJIF Impact Factor Volume 4, Issue 10, Research Article ISSN EVALUATION OF INVITRO ANTIUROLITHIATIC ACTIVITY OF VIDANGADI CHURNA Sajith Mohandas 1*, Shalveena Nizar, Radhika.P.V Rajiv Gandhi Institute of Pharmacy, Trikaripur, Kasargod, Kerala Article Received on 28 July 2015, Revised on 17 Aug 2015, Accepted on 06 Sep 2015 *Correspondence for Author Asst. Prof. Sajith Mohandas Rajiv Gandhi Institute of Pharmacy, Trikaripur, Kasargod, Kerala ABSTRACT activity, Calcium oxalate, Nucleation. Objective: Vidangadi churna is an ayurvedic formulation, was investigate for antiurolithiatic activity. Method: Calcium oxalate crystallization was induced by the addition of 0.01M sodium oxalate solutions in synthetic urine and nucleation method. Result: The effect of Vidangadi churna exhibited a concentration dependent inhibition of calcium oxalate crystallization and nucleation. Conclusion: The present studies suggest that Vidangadi churna has a potential inhibition of calcium oxalate crystallization exhibited and nucleation. Vidangadi Churna showed potent antiurolethiatic activity. KEYWORDS: Vidangadi churna, urolithiasis, Antiurolethiatic INTRODUCTION In developing countries all over the world, 80% of population continues to use traditional medicine in primary medical problems. In the past decade, therefore, research has been focused on scientific evaluation of traditional drugs of plant origin for primary medical problems. Herbal medicines have several phytoconstituents and exert their beneficial effects on urolithiasis or kidney stones by multiple mechanisms. Urolithiasis (nephrolithiasis) or kidney stone is formation of urinary calculi at any level of urinary tract. It is estimated that 12% of world population experiences renal stone disease with a recurrence rate of 70%-80% in male and 47%-60% in female. [1, 2] Kidney stone formation or urolithiasis is a complex process that is a consequence of an imbalance between promoters and inhibitors in the kidneys. The recurrence of urolithiasis represents a serious problem as patients who have formed one stone are more likely to form another. [3] The pathogenesis of urolihiasis seems to be mulifacorial and complicated. [4] Most calculi in the urinary system arise from a common Vol 4, Issue 10,

2 compartment of urine, e.g., calcium oxalate (CaOx), representing upto 80% of analyzed stones. [5] A number of medicinal plants have been used in India and elsewhere which claim efficient cure of urinary stones. [6] Unfortunately, the underling risk factors are not corrected by these techniques; hence there is a need to continue medical supervision and therapy to prevent stone recurrence. [7] Vidangadi churna is one of the traditional polyherbal preparations, composed of Embelia ribes (false black pepper fruit), Hordeum vulgare, Mallotus philippinensis, Terminalia chebula (fruit rind) and rock salt. Embelia ribes Burm F., a medicinal woody climber, belongs to the Myrsinaceae family. [8] Terminalia chebula locally named as harad in India has been extensively used in ayurveda. It is used to treat urolithiasis and is actively used in various drug formulations of kidney stone treatments. It is extensively explored for antimicrobial. [9] antioxidant. [10] anticarcinogenic. [11] hypocholesterolemic. [12] and diuretic. [11, 12] activities by various research groups. It is used in the preparation of Vaishvanara churna which is an ayurvedic formulation used in the treatment of arthritis, constipation, abdominal pain and improves digestion, strength and immunity. The studies on this churna show antiurolithiatic activity. [13] The main aim of the present investigation was to screen antiurolithiatic activity of Vidangadi churna. MATERIALS AND METHODS Vidangadi churna contains Embelia ribes, Hordeum vulgare, Mallotus philippinensis, Terminalia chebula and rock salt were made in to fine powder and passed through 100 No. sieve and mixed. Vidangadi churna was extracted with water for antiurolethiatic study. EXPERIMENT PROTOCOL Invitro Antiurolethiatic Activity 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 0.01M 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. Preparation of Artificial Urine The artificial urine (AU) was prepared according to the method Burns and Finlayson. [14] with slight modification and the following composition: sodium chloride mm, sodium phosphate 32.3 mm, sodium citrate 3.21 mm, magnesium sulfate 3.85 mm, sodium sulfate Vol 4, Issue 10,

3 16.95 mm, potassium chloride 63.7 mm, calcium chloride 4.5 mm, sodium oxalate 0.32 mm, ammonium hydroxide 17.9 mm, and ammonium chloride mm. The AU was prepared fresh each time and ph adjusted to 6.0. Study without inhibitor A volume of 1.0 ml of AU was transferred into the cell and 0.5 ml of distilled water added to it and blank reading was taken. The 0.5 ml of 0.01M sodium oxalate was added, to the previous volume, and the measurement is immediately started for a period of ten minutes. Study with inhibitor The aqueous extract of Vidangadi churna was dissolved in distilled water, filtered through membrane filter and the concentrations of 100, 200, 400, 600, 800 and 1000 μg/ml was obtained. A mixture of 1 ml of AU and 0.5 ml of churna extract solution is versed in the cell. A blank reading was taken and then 0.5 ml of 0.01M sodium oxalate solution was added and immediately the absorbance was measured for a period of ten minutes at 620nm. [15] The percentage of inhibition of calcium oxalate crystal formation was calculated using the following formula: Where; Ab Test: Absorbance in the presence of inhibitor (Extract), Ab Control: Absorbance without inhibitor (Control). Nucleation Assay The method used was similar to that described by Hennequin et al. [16] with slight modifications. Solutions of calcium chloride and sodium oxalate were prepared at the final concentration of 3 mm and 0.5 mm, respectively, in a buffer containing Tris 0.05 M and NaCl 0.15 M at ph 6.5. Both solutions were filtered through a 0.22 μm filter; 33 ml of calcium chloride solution was mixed with that of the aqueous extract of Vidangadi churna of different concentrations. Crystallization was started by adding 33 ml of sodium oxalate solution. The final solution was magnetically stirred at 800 rpm. The temperature was maintained at 37 C. The absorbance of the solution was monitored at 620 nm. The percentage inhibition produced by the herb extract was calculated as: Vol 4, Issue 10,

4 Where; Ab Test: Absorbance in the presence of inhibitor (Extract), Ab Control: Absorbance without inhibitor (Control). RESULTS Figure 1 showed the graph of percentage inhibition of the crystallization of calcium oxalate (CaOx) with different concentrations of aqueous extract of Vidangadi churna. It inhibited the crystallization in a concentration dependent pattern. The percent inhibition was calculated using the above mentioned formula. The percentage inhibition shown by aqueous extract at 100 μg/ml was 76.2 % and with almost constant inhibition at 200 μg/ml and 1000 μg/ml in the range of % Fig.1 Effect of different concentrations of extract of Vidangadi churna on calcium oxalate crystallization. Figure 2 displays the effect of the different concentration of the aqueous extract of Vidangadi churna on the nucleation of calcium oxalate crystals. The increase in the concentration of Vidangadi churna extract showed increase in the inhibition of nucleation. Maximum inhibition of nucleation 100 % observed at concentration of 2000 μg/ml. Vol 4, Issue 10,

5 Fig.2 Effect of aqueous extract of Vidangadi churna on nucleation of calcium oxalate. DISCUSSION There has been a long standing quest for potent inhibitors of calcium oxalate growth as it is the most common urinary stone associated with renal injury. Recent evidence suggests that in many calcium oxalate stone formers the earliest changes may be calcium salt deposition in the medullary interstitium. In marked hyperoxaluric states, primary hyperoxaluria directs calcium oxalate crystal adhesion to renal epithelial cells. [17] Calcium oxalate stones are found in two different varieties, calcium oxalate monohydrate (COM) or Whewellite, and calcium oxalate dihydrate (COD) or Weddellite. COM, the thermodynamically most stable form, is observed more frequently in clinical stones than COD and it has a greater affinity for renal tubular cells, thus responsible for the formation of stones in the kidney. [18] In the present study, the anticalcifying properties of Vidangadi churna were explored by in vitro. After nucleation, crystal growth is the next major step of stone formation. The driving force for crystallization is a reduction in the potential energy of the atoms or molecules when they form bonds to each other. The crystal growth process starts with the nucleation stage when several atoms or molecules in a supersaturated liquid start to form clusters. [19] Nucleation is the formation of a solid crystal phase in a solution. It is an essential step in renal stone formation. [20, 21] The inhibitory potency of the Vidangadi churna was tested on the nucleation and growth of the most commonly occurring kidney stones, calcium oxalate monohydrate. A concentration dependent inhibition was observed using Vidangadi churna. Further the invitro antiurolithiatic activity of Vidangadi churna is mainly due to the presence Vol 4, Issue 10,

6 of Terminalia chebula which was proved by earlier studies to contain cytoprotective properties towards the MDCK (Madin Darby Canine Kidney cells) and NRK-52E cells (Normal Rat Kidney Epithelial cells) by reducing the LDH (Lactate Dehydrogenase) leakage and increasing the cell viability and inhibit the calcium oxalate crystals in vitro. [22] The churna also contains Hordeum vulgare which possess diuretic and urinary antiseptic properties. [23] Thus these synergetic effects of Vidangadi churna show invitro antiurolithiatic effect. CONCLUSION The present study provides a scientific proof of Vidangadi churna for invitro antiurolithiatic activity. Hence further in vivo studies are required to support this claim. ACKNOWLEDGEMENT The authors are grateful to The Chairman, The Principal and the Management of Rajiv Gandhi Institute of Pharmacy, Trikaripur, Kerala, India for providing necessary facilities to carry out the research work. REFERENCES 1. Parmar RK, Kachchi NR, Tirgar PR, Bhalodiya PN. Preclinical evaluation of Antiurolithiacic activity of Swertia chirata Stems. Int Res J Pharm, 2012; 3(8): Sundararajan P, Mahesh R, Ramesh T, Hajeena B. Effect of Aervalanataon calcium oxalate urolithiasis. Indian J Asian Boil, 2006; 24: Khan NI, Shinge JS, Naikwade NS. Antilithiatic effect of Helianthus annuus Linn. Leaf extract in ethylene glycol and ammonium chloride induced nephrolithiasis. Int J Pharm Sci, 2010; 2(4): Lee,Y., W. Huang, J. Huang and L. Chang, Testosterone Enhances whereas Estrogen inhibits calcium oxalate stone formation in Ethylene Glycol Treated Rats. J. Urol., 1991; 156: Prien, E.L. and E.L.J. Prien, Composition and structure of urinary stone. Am.J. Med., 1968; 45: Mukharjee, T., N. Bhalla, G.S. Aulah and H.C.Jain, Herbal drugs for urinary stones literature appraisal. Indian Drugs, 1984; 21: Halabe, A. and O. Sperling, Uric acid nephrolithiasis. Miner Electrolyte Metab., 1994; 20: Vol 4, Issue 10,

7 8. Syed Asadulla, Ramandang and Rajasekharan Pharmacognosy of Embelia Ribes Burm F International Journal of Research in Pharmacy and Chemistry, 2011; 1: Cheng HY, Lin TC, Yu KH, Yang CM, Lin CC: Antioxidant and free radical scavenging activities of Terminalia chebula. Biol Pharm Bull, 2003; 26: Saleem A, Husheem M, Härkönen P, Pihlaja K: Inhibition of cancer cell growth by crude extract and the phenolics of Terminalia chebula retz. fruit. J Ethnopharmacol, 2002; 81: Thakur CP, Thakur B, Singh S, Sinha PK, Sinha SK: The Ayurvedic medicines Haritaki, Amala and Bahira reduce cholesterol-induced atherosclerosis in rabbits. Int J Cardiol,1988; 21: Chattopadhyay RR, Bhattacharyya SK: Terminalia chebula: An update. Phcog Rev, 2007; 1: Ashok Kumar, B.S., Saran, G., Harshada, R., Keerthi, N., Vandana, S., Evaluation of invitro antiurolithiatic activity of Vaishvanara churna. Journal of Medicinal Plants studies, 2013; 1(3): Burns JR, Finlayson B. Changes in calcium oxalate crystal, morphology as a function of concentration. Invest Urol, 1980; 18(2): Bensatal A, Ouahrani MR. Inhibition of crystallization of calcium oxalate by the extraction of Tamarix gallica L. Urol Res, 2008; 36(6): Hennequin C, Lalanne V, Daudon M, Lacour B, Drueke T. A new approach to studying inhibitors of calcium oxalate crystal growth. Urol Res, 1993; 21(2): Atmani F, Farell G, Lieske JC: Extract from Herniaria hirsute coats calcium oxalate monohydrate crystals and blocks their adhesion to renal epithelial cells. J Urol, 2004; 172: Verkoelen CF, Romijn JC, De Bruijn WC, Boeve ER, Cao LC, Schroder FH. Association of calcium oxalate monohydrate crystals with MDCK cells. Kidney Int, 1995; 48(1): Qiu SR, Wierzbicki A, Orme CA, Cody AM, Hoyer JR, Nancollas GH, et al. Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. Proc Natl Acad Sci U S A, 2004; 101(7): Finlayson B. Physicochemical aspects of urolithiasis. Kidney Int, 1978; 13(5): Khan SR, Byer KJ, Thamilselvan S, Hackett RL, McCormack WT, Benson NA, et al. Crystal-cell interaction and apoptosis in oxalate-associated injury of renal epithelial cells. J Am Soc Nephrol, 1990; 10(14): S Vol 4, Issue 10,

8 22. Tayal.S, Duggal.S, Bandyopadhyay.P, Aggarwal.A, Tandon.S, and Tandon.C, Cytoprotective role of the aqueous extract of Terminalia chebula on renal epithelial cell. 23. Katha, S., Natural Benefits and Curative Properties of Barley. Available at: regional of kerala.blogspot.com/2011/07/ natural- benefits-and-curative recipes.html Retrieved May 7. Vol 4, Issue 10,