|Year : 2012 | Volume
| Issue : 3 | Page : 429-434
Evaluation of anti-urolithiatic activity of Pashanabhedadi Ghrita against experimentally induced renal calculi in rats
Sanjay Kumar Gupta1, Madhav Singh Baghel2, Chaturbhuja Bhuyan3, B Ravishankar4, BK Ashok5, Panchakshari D Patil6
1 Associate Professor and I/C Head, Department of Shalya Tantra, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
2 Director, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Director, Centre for Care of Ano-Rectal Research by Indian System of Medicine and Allied Science, Bhubaneshwar, Orissa, India
4 Director, Research and Development, SDM Ayurveda College, Kuthpady, Udupi, Karnataka, India
5 Research Associate, Drug Discovery Group, Research and Development Himalaya Health Care, Bengaluru, Karnataka, India
6 Senior Research Fellow, Institute for Post Graduate Teaching and Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
|Date of Web Publication||16-Mar-2013|
Sanjay Kumar Gupta
Department of Shalya Tantra, IPGT and RA, Gujarat Ayurved University, Jamnagar- 361 008, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Population in an industrialized world is afflicted by urinary stone disease. Kidney stones are common in all kinds of urolithiasis. One distinguished formulation mentioned by Sushruta for management of Ashmari (urolithiasis) is Pashanabhedadi Ghrita (PBG), which is in clinical practice since centuries. Validation of drug is the requirement of time through the experimental study. In this study, trial of PBG has been made against ammonium oxalate rich diet and gentamicin injection induced renal calculi in albino rats. The calculi were induced by gentamicin injection and ammonium oxalate rich diet. Test drug was administered concomitantly in the dose of 900 mg/kg for 15 consecutive days. Rats were sacrificed on the 16 th day. Parameters like kidney weight, serum biochemical, kidney tissue and histopathology of kidney were studied. Concomitant treatment of PBG attenuates blood biochemical parameters non-significantly, where as it significantly attenuated lipid peroxidation and enhanced glutathione and glutathione peroxidase activities. It also decreased crystal deposition markedly into the renal tubules in number as well as size and prevented damage to the renal tubules. The findings showed that PBG is having significant anti-urolithiatic activities against ammonium oxalate rich diet plus gentamicine injection induced urolithiasis in rats.
Keywords: Ammonium oxalate, Ashmari, gentamicin, Pashanabhedadi Ghrita, urolithiasis
|How to cite this article:|
Gupta SK, Baghel MS, Bhuyan C, Ravishankar B, Ashok B K, Patil PD. Evaluation of anti-urolithiatic activity of Pashanabhedadi Ghrita against experimentally induced renal calculi in rats. AYU 2012;33:429-34
|How to cite this URL:|
Gupta SK, Baghel MS, Bhuyan C, Ravishankar B, Ashok B K, Patil PD. Evaluation of anti-urolithiatic activity of Pashanabhedadi Ghrita against experimentally induced renal calculi in rats. AYU [serial online] 2012 [cited 2021 Jul 30];33:429-34. Available from: https://www.ayujournal.org/text.asp?2012/33/3/429/108860
| Introduction|| |
Mankind has been afflicted by urinary stones (Urolithiasis) since centuries, and it is proven to be an important cause of renal failure. Even in the 4 th century B.C., Hippocrates is noted the presences of the renal stone together with renal abscess and he has mentioned that in his Hippocratic oath "… I will not cut, even for stone, but leave such procedures to the practitioners of the craft. " The specialty of urology branch has been recognized since ever. It is estimated that at least 10% of the population in the industrialized part of the world is afflicted by urinary tract stone disease. Among those, kidney stones are common in industrialized nations with an annual incidence of 0.5-1.9%. , About 12% of the population of India is expected to have urinary stones and out of that about 50% of cases encounter loss of one or both kidneys with or without renal damage upto some extent. Nearly 15% of the population of northern India is also suffering from kidney stones. Upper as well as lower urinary tract stones occur frequently but the incidence shows wide variation on the regional basis in India. 
Ayurveda, a traditional system of Indian medicine, recommends several medicinal plants and compound medicinal preparations for the treatment of urolithiasis. , Herbs and herbal drugs have created interest among the people by its clinically proven effects. The overuse of synthetic drugs, results in higher incidence of adverse drug reactions, has motivated humans to return to nature for safe remedies. One such distinguished formulation mentioned by Sushruta in the management of Ashmari (urinary stones) is Pashanabhedadi Ghrita (PBG)  which is more popular among Vaidyas (physicians). The name of the formulation itself suggests anti-urolithiatic action, i.e., Pashana means stone and Bheda means to crush. However, until date there is no experimental basis is available to prove clinical evidence on this formulation, hence the present study was designed to evaluate anti-urolithiatic activity of PBG in experimental animals.
| Materials and Methods|| |
Wistar strain albino rats of either sex; weighing 160-220 g were used for the study. The animals were obtained from the animal house attached to our institute. Six animals were housed in each cage made up of poly-propylene with stainless steel top grill. The dry wheat (post-hulled) waste was used as bedding material and was changed every morning. The animals were exposed to 12-h light and 12 h dark cycle with the relative humidity of 50-70% and the ambient temperature during the period of experimentation was 22 ± 03°C. Animals were fed with Amrut brand rat pellet feed supplied by Pranav Agro Mills Pvt. Limited. Tap water ad libitum was used for their drinking purpose. The experiments were carried out in conformity with the Institutional Animal Ethics Committee (IAEC) after obtaining its permission (IAEC03/08-11/PhD/01).
The raw materials [Table 1] of the test formulation were procured from pharmacy attached to Gujarat Ayurved University, Jamnagar and authenticated by pharmacognosist. From these raw drugs, "PBG" was prepared in the Department of Rasashastra and Bhaishajya Kalpana of I.P.G.T. and R.A. as per the classical reference of Ghrita Paka Kalpana.  The finished product was stored in air tight glass container and utilized for experimental study.
The human dose of PBG mentioned in classical texts is 10 g per day. Considering this, the dose of the experimental animals was calculated by extrapolating the human dose to rat dose as 900 mg/kg based on the body surface area ratio by referring to the standard table of Paget and Barnes (1964).  The test drug was administered orally with the help of a gastric catheter of suitable size sleeved onto a disposable syringe.
Hyperoxaluria and calcium oxalate deposition in the kidney was induced by using injection gentamicin (40 mg/kg/Intra Peritonial) and Calculi Producing Diet (CPD).  The diet is made by powdered standard rat pellet feed mixed with ammonium oxalate (5%), then made into pellet and dried up properly. The selected animals were weighed and randomly divided into four groups, consisting of six animals in each group. Group-I received only distilled water orally for 15 days, served as normal control. Group-II received gentamicin (40 mg/kg, Sub-cutaneous., day 1 st -8 th ), CPD and water (from day 1 st to 15 th ) and served as negative control (diet control). Group-III received gentamicin (40 mg/ kg, SC., day 1 st -8 th ); CPD and plain Goghrita (0.9 g/kg, orally, day 1 st -15 th ) and served as Vehicle Control (VC). Group-IV received gentamicin (40 mg/kg, SC., day 1 st ?8-8 th ); CPD and Pashanabhedadi Ghrita (0.9 g/kg, orally, day 1 st -15 th ) and coded as PBG. The gentamicin injection was given after 2 h of drug administration, and normal diet was replaced by CPD for 15 days.
Assessment of urinary parameters
On the 15 th day after-drug administration, the rats of all the four groups were hydrated with distilled water (2 ml/100 g rat), housed in separate metabolic cages and urine samples were collected for 24 h and the urinary pH and specific gravity were determined by strip method.
Assessment of kidney parameters
On the 16 th day the animals were weighed and anaesthetized by diethyl ether. Blood was collected from retro orbital plexus by capillary puncturing for estimation of serum biochemical parameters such as serum urea,  serum creatinine,  serum uric acid,  and serum calcium.  Then the animals were sacrificed by over dose of ether anesthesia. The abdomen was opened by midline incision and kidney was dissected out carefully and cleaned off the extraneous tissue. Kidney was weighed and one kidney of each animal was transferred to 10% formalin solution for the purpose of histopathological studies while the other kidney was utilized for estimation of biochemical parameters in tissue homogenate parameters. The parameters such as lipid peroxidation,  nitric oxide,  total glutathione,  and glutathione peroxides  activities were estimated as per standard procedures. The histopathological slides were prepared by referring standard procedure.  The slides were viewed under binocular research Carl-Zeiss's microscope (Germany) at various magnifications to note down the changes in the microscopic features of the tissues studied.
The obtained data have been presented as mean ± SEM, difference between the groups was statistically determined by Student t-test for unpaired data with the level of significance set at P < 0.05.
| Results|| |
An apparent and statistically significant increase in weight of kidney was occurred in the diet control group in comparison to the normal control group. Administration of Goghrita and PBG failed to attenuate the weight of kidney to a significant extent in comparison to diet control group [Table 2]. Feeding of ammonium oxalate diet leads to significant decrease in urinary pH in comparison to normal control. Administration of PBG non-significantly attenuated pH of urine.
A statistically significant increase in blood urea and serum creatinine level was occurred by feeding of ammonium oxalate rich diet. Further marked elevation of uric acid level was also noted in comparison to the normal control group. Treatment with PBG failed to attenuate these factors to a significant extent [Table 3].
Feeding of ammonium oxalate rich diet for 15 days along with gentamicin injection leads to significant increase in lipid peroxidation and nitric oxide levels in kidney tissue homogenate in comparison to the normal control group. Treatment with PBG significantly attenuated lipid peroxidation. Further, administration of plain ghee (Vehicle) and PBG significantly enhanced the level of total glutathione and glutathione peroxidase activity [Table 4].
|Table 4: Effect on tissue biochemical parameters of kidney tissue homogenate|
Click here to view
Microscopic examination of kidney sections from the normal control group showed normal cytoarchitecture [Figure 1]. Sections from gentamicin injection plus ammonium oxalate diet control group shows the presence of a large number of crystal material containing tubules, especially in the cortical region; dilatation of tubules (due to stones) along with necrosis of the tubular epithelium [Figure 2]. Sections from VC treated group shows a moderate decrease in the size and number of crystal containing tubules and mild tubular epithelial necrosis [Figure 3], while marked decrease of these features are observed in PBG treated group [Figure 4].
|Figure 1: Photomicrograph of representative sections of kidney of albino rats from control group (1 × 400 magnification). Ct: Convoluted tubule. Note: Normal cytoarchitecture|
Click here to view
|Figure 2: Photomicrograph of representative sections of kidney of albino rats from ammonium oxalate (diet) control group (1 × 400 magnification). Note: Presence of large number of crystal material containing tubules especially in the cortical region (Arrow head)|
Click here to view
|Figure 3: Photomicrograph of representative sections of kidney of albino rats from vehicle (Plain Ghrita) control group (1 × 400 magnification). Note: Marked decrease in the size and number of crystal containing tubules|
Click here to view
|Figure 4: Photomicrograph of representative sections of kidney of albino rats from Pashanabhedadi Ghrita treated group (1 × 400 magnification). Note: Marked decrease in the size and number of crystal containing tubules|
Click here to view
| Discussion|| |
The pathogenesis of calcium oxalate (CaOx) stone formation is a multi-step process and in essence includes urinary saturation, urinary super saturation, nucleation, crystal growth, crystal aggregation, and crystal retention. Various substances in the body have an effect on one or more of the above stone forming processes and thereby influencing a person's ability of the body to promote or prevent stone formation. Promoters of stone formation facilitate stone formation whilst inhibitors prevent it. Low urine volume, low urine pH, calcium, sodium, oxalate, and urate are known to promote stone formation. Many inorganic (e.g., citrate, magnesium) and organic (e.g., urinary prothrombin fragment 1, glycosaminoglycans, osteopontin) substances are known to inhibit stone formation. Organic inhibitory compounds adsorb to the surface of the crystals, thereby inhibiting crystal growth and nucleation. 
The incidence and nature of spontaneous urolithiasis are imperfectly known in the rats.  In the present study, 5% ammonium oxalate is used instead of 3% ammonium oxalate as reported by Sanjay Kumar et al., because the preliminary studies have shown less incidence of calculi deposition with 3% ammonium oxalate. This treatment schedule of gentamicin and ammonium oxalate rich diet increases calcium and oxalate super saturation, renal tubular injury and produce conditions conducive to the formation and growth of CaOx stones.  The main causes of CaOx stone formation appear to be chronic hyperoxaluria.  In the present study, gentamicin and ammonium oxalate rich diet-induced hyperoxaluria not only increased CaOx deposition in the kidney but also causes papillary damage and incrustations as reported earlier.  This treatment schedule of gentamicin and ammonium oxalate increase calcium and oxalate super saturation, renal tubular injury and produce favourable condition to the formation and growth of CaOx stones. 
In pondral changes, it is observed that significant increase in kidney weight of the negative control group. This increase supported the results of stone deposition in kidney; it was also supported by histopathological study in which plenty of stones were present in sections of kidney of this group [Figure 2]. Further, extensive degenerative changes are also observed in kidneys of this group, but administration of PBG and VC in animals was failed to attenuate increased kidney weight in comparison to the negative control group.
The type of stones formed can be predicted from the pH of the fasting urine. Crystalluria is pH dependant. Dissolution of calculi can be achieved by alteration in urinary pH form If the pH is acidic 5.5 or below, the stones are likely to be of acidic type (uric acid),  if 5.0-6.5 CaOx  type and if alkaline (7.2 or above) indicates magnesium ammonium phosphate type. In the present study a decrease in pH was observed on induction of ammonium oxalate type of stones in negative control. Treatment with PBG reversed acidic pH to slightly alkaline pH (7.2 ± 0.20), where as in VC group pH of urine was observed neutral (7.0 ± 0.25). This increase in urinary pH might be responsible for dissolving the complexes of ammonium and oxalate. Thus, some of the anti-urolithiatic effects of PBG and VC are possibly due to its effect on urinary pH.
Due to the presence of stones, there is obstruction to the out flow of urine and because of this, the Glomerular Filtration Rate (GFR) also decreases. Reduction in the GFR leads to accumulation of the waste products, particularly nitrogenous substances such as urea, creatinine, and uric acid in blood.  Blood urea nitrogen level is considered as a good indicator of balance in the nitrogen metabolism. It tends to enhance with increase tissue catabolism. In the present study significant increase in blood urea level was observed in the negative control group where as in the treated group with PBG and VC failed to attenuate it. Enhanced serum creatinine indicates renal impairment due to hyperoxaluria. Treatment with PBG and VC failed to attenuate elevated serum creatinine level reflected impaired renal function. Other serum biochemical parameters (serum uric acid and calcium) were not affected to a significant extent by ammonium oxalate rich diet plus gentamicin administration in any group.
Studies show that oxalate, a major stone-forming constituent, has been reported to induce lipid peroxidation and causes tissue damage by reacting with polyunsaturated fatty acids in cell membranes.  The polyunsaturated fatty acid content of kidney makes it prone to Reactive Oxygen Species (ROS) attack.  Imbalance between oxidants and anti-oxidants level result in Oxidative Stress (OS). Low levels of renal cellular glutathione are reported to favor lipid peroxidation and retain calcium and oxalate in the kidney.  In the present study, it is observed that ammonium oxalate rich diet, which was fed to rats with injection gentamicin leads to significant increase in lipid peroxidation level in kidney tissue of the negative control group in comparison to normal control. But in PBG group, it is observed that treatment with PBG significantly attenuated lipid peroxidation. Hence PBG may be considered to prevent the lipid peroxidation-induced renal damage caused by CaOx crystal deposition in the kidney tissue. Therefore, PBG can be attributed to check CaOx crystal attachment and stones formation.
The impaired anti-oxidant protection might be responsible for the accumulation and retention of oxalate and subsequent deposition of CaOx in the kidney. Treatment with anti-oxidants was reported to reduce hyperoxaluria and the resultant OS in rats.  In present study ammonium oxalate rich diet with gentamicin administration significantly decreased glutathione and glutathione peroxidation activity and non-significantly increased nitric oxide level in negative control group. Treatment with PBG and VC decreased nitric oxide level non-significantly and enhanced anti-oxidant system like glutathione peroxidase and total glutathione content significantly. This shows anti-urolithiatic effects of PBG and VC, which might be due to quenching oxidant constituents in the kidney. It was reported that the protective effect of Berginia ligulata in hyperoxaluric OS and CaOx crystal deposition is due to their potential anti-oxidant activity. Achyranthus aspera, Quercus salieina, Amni visnaga, and Mimusops elengi, which are also ingredients of test formulation are also reported to be having the protective effect against oxalate-induced renal tubular epithelial cell injury in cell culture due to their anti-oxidant activity.  Further one more ingredient of this formulation, Varuna (Crataeva nurvala) prevents stone formation due to the anti-lithogenic activity and the anti-crystallization property.  It was also reported that Crateva nurvala, Tribulus terrestris, and Dolichos biflorus were found to be effective in preventing the deposition of the stones in experimental rats.  The ethanolic extract of Asparagus racemosus Wild had an inhibitory potential on lithiasis induced by oral administration significantly reduced the elevated level of calculogenic ions in urine and it elevated the urinary concentration of magnesium, which is considered as one of the inhibitors of crystallization.  Thus, the observed anti-urolithiatic activity of test formulation in the present study may be attributed to collective effect of these drugs. The mechanism involved in observed activity profile may be,
- Improving the renal tissue anti-oxidant status and cell membrane integrity
- Inhibition of crystal nucleation, aggregation and growth,
- By increasing urine volume, pH and anti-calcifying activity,
- Regulation of oxalate metabolism.
| Conclusion|| |
PBG is having significant anti-urolithiatic activity besides it is having marked anti-oxidant activity. However, further detailed study is required to explore the active principle responsible for this and also to know the exact mechanism involved in observed activity profile.
| References|| |
|1.||Stamatelou KK, Francis ME, Jones CA, Nyberg LM, Curhan GC. Time trends in reported prevalence of kidney stones in the United States: 1976-1994. Kidney Int 2003;63:1817-23. |
|2.||Lieske JC, Pena de la Vega LS, Slezak JM, Bergstralh EJ, Leibson CL, Ho KL, et al. Renal stone epidemiology in Rochester, Minnesota: An update. Kidney Int 2006;69:760-4. |
|3.||Colobawalla BN. Incidence of urolithiasis in India. ICMR Tech Rep 1971;8:42-51. |
|4.||Agarwal S, Gupta SJ, Saxena AK, Gupta N, Agarwal S. Urolithic property of Varuna (Crataeva nurvala): An experimental study. AYU 2010;31:361-6. |
|5.||Singh RG, Behura SK, Kumar R. Litholytic property of Kulattha (Dolichous biflorus) vs potassium citrate in renal calculus disease: A comparative study. J Assoc Physicians India 2010;58:286-9. |
|6.||Susruta, Susruta Samhita, Kaviraj Ambikadatta Shastri editor. 12 th ed. Chaukhambha Sanskrit Sansthan, Varanasi; 2001. Chikitsa Sthana, Ashmari Chikitsa Adhyaya, 7/5-8 p. 41. |
|7.||Sharangdharacharya, Sharangdhara Samhita. Commentator, Dr. Brahmananda Tripathi. Chaukhambha Sanskrit Surabharati Prakashana, Varanasi; 2008. Madhyam Khanda, Snehakalpana 9/1-2.p. 218. |
|8.||Paget GE, Barnes JM. Toxixity Tests. In: Laurence DR, Bacharach AL, editors. Evaluation of drug activities pharmacometrics. New York: Academic Press; 1964. p. 161. |
|9.||Kumar S, Sigmon D, Miller T, Carpenter B, Khan S, Malhotra R, et al. A new model of nephrolithiasis involving tubular dysfunction/injury. J Urol 1991;146:1384-9. |
|10.||Tiffany TO, Jansen JM, Burtis CA, Overton JB, Scott CD. Enzymatic kinetic rate and end-point analyses of substrate, by use of a GeMSAEC fast analyzer. Clin Chem 1972;18:829-40. |
|11.||Slot C. Plasma creatinine determination. A new and specific Jaffe reaction method. Scand J Clin Lab Invest 1965;17:381-7. |
|12.||Kabasakalian P, Kalliney S, Westcott A. Determination of uric acid in serum, with use of uricase and a tribromophenol-aminoantipyrine chromogen. Clin Chem 1973;19:522-4. |
|13.||Tietz NW. Textbook of Clinical Chemistry. W. B. Saunders; 1986. p. 1350. |
|14.||Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8. |
|15.||Sreejayan N, Rao MN. Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol 1997;49:105-7. |
|16.||Grunert RR, Phillips PH. A modification of the nitroprusside method of analysis for glutathione. Arch Biochem 1951;30:217-25. |
|17.||Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG. Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90. |
|18.||Raghuramulu N, Nair KM, Kalyanasundaram S. A Manual of Laboratory Techniques. 1 st ed. Hyderabad: National Institute of Nutrition; 1983. p. 246-53. |
|19.||Doddametikurke RB, Biyani CS, Anthony JB, Jon JC. The role of urinary kidney stone inhibitors and promoters in the pathogenesis of calcium containing renal stones. EAU-EBU Update Ser 2007;5:93-138. |
|20.||Magnusson G, Ramsay CH. Urolithiasis in the rat. Lab Anim 1971;5:153-62. |
|21.||Khan SR. Animal models of kidney stone formation: An analysis. World J Urol 1997;15:236-43. |
|22.||Baumann JM. Stone prevention: Why so little progress? Urol Res 1998;26:77-81. |
|23.||Khan SR. Hyperoxaluria-induced oxidative stress and antioxidants for renal protection. Urol Res 2005;33:349-57. |
|24.||Guilherme AA, Fernanda TB, Nestor S. Uric acid and renal function. In: Sahay M, editor. Diseases of renal parenchyma. In Tech; 2012. pp. 57-74. Available from: http://www.intechopen.com/books/diseases-of- renal-parenchyma/uric-acid-and-renal-function [Last accessed 2012 Mar 30]. |
|25.||Puotinen CJ, Straus M. Treatment and prevention of calcium oxalate kidney and bladder stones. Whole Dog J 2010;5. Available from: http://www.dogaware.com/articles/wdjcalciumoxalates.html [Last accessed 2012 Mar 30]. |
|26.||Ashok P, Koti BC, Vishwanathswamy AH. Antiurolithiatic and antioxidant activity of Mimusops elengi on ethylene glycol-induced urolithiasis in rats. Indian J Pharmacol 2010;42:380-3. |
|27.||Devinder S, Rajnendrapal K, Vikas C, Kanwaljit C. Mini-review: Antioxidants in the prevention of renal disease. J Med Food 2006;9:443-50. |
|28.||Toblli JE, Ferder L, Stella I, De Cavanaugh EM, Angerosa M, Inserra F. Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria. J Urol 2002;168:1550-5. |
|29.||Thamilselvan S, Byer KJ, Hackett RL, Khan SR. Free radical scavengers, catalase and superoxide dismutase provide protection from oxalate-associated injury to LLC-PK1 and MDCK cells. J Urol 2000;164:224-9. |
|30.||Yadav RD, Jain SK, Shashi A, Mahor A, Bharti JP, Jaiswal M. A review: Herbal plants used in the treatment of Urolithiasis. Int J Pharm Sci Res 2011;2:1412-20. |
|31.||Agarwal S, Gupta SJ, Saxena AK, Gupta N, Agarwal S. Urolithic property of Varuna (Crataeva nurvala): An experimental study. AYU 2010;31:361-6. |
|32.||Kumar P, Despande PJ, Singh LM. Kidney stone dissolving drugs. Bull Med Ethanobot Res 1981;2:277-84. |
|33.||Alvin JM, Ibrahim S, Janardhan. Modulatory effect of Plectranthus amboinicus Lour on ethylene glycol induced nephrolithiasis in rats. Indian J Pharmacol 2005;37 : 43-5 |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]