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PROPHYLACTIC EFFECT OF ‘NANDUKKAL PARPAM’ (A SIDDHA COMBINATION DRUG) ON ETHYLENE GLYCOL INDUCED CALCIUM OXALATE MICROLITHIASIS IN THE KIDNEYS OF WISTAR RATS
N ARUNAI NAMBIRAJ*, TMR PANICKER**, S SEETHALAKSHMI***,CHINNAMA ABRAHAM+, M PAUL KORATH++, K JAGADEESAN+++
*Research Associate; **Research Officer; ***Professor and Head of the Department of Laboratory Medicine; +Chief Medical Technologist; ++Chief Physician/Nuclear Cardiologist; +++Director, KJ Hospital Research and Post Graduate Centre, Chennai 600 084.
The effect of oral administration of ‘Nandukkal parpam’ (a siddha combination drug) on calcium oxalate microlithiasis was studied in male wistar rats. Ethylene glycol and ammonium chloride in drinking water were given orally to male wistar rats to induce calcium oxalate crystals in renal tissue the initial phase of urinary stone formation. The deposition of calcium oxalate crystals in kidneys of wistar rats on ethylene glycol and treated with nandukkal parpam is much lesser than in the group of rats on ethylene glycol only (p < 0.001).
INTRODUCTION
A large number of individual herbal drugs and their combinations have been used in Siddha and Ayurveda systems of medicine from time immemorial against urinary stone disease in humans. It is mentioned in the siddha text, that ‘Nandukkal parpam’ a combination drug consisting of five constitutents, one of which is Nandukkal is reported to have anti urinary calculus properties. Earlier work done in this institution[1] and many other references in literature[2-4] have shown that out of the three most common constituents of human urinary stones namely calcium oxalate, calcium phosphate and magnesium ammonium phosphate hexahydrate, calcium oxalate is present in about three-fourths of all kidney stones. The present study is a preliminary investigation to understand the prophylactic role of ‘Nandukkal parpam’ when administered orally to male wistar rats in whom calcium oxalate crystals were induced in the kidneys.
MATERIAL AND METHODS
Constituents of Nandukkal parpam
The combination siddha drug has the following constituents as described in the Formulary of Siddha Medicines.[5]
Water of lime (from lime stones)
Kalchunnambu neer
Solution of alkaline (earth salts)
Pooneeru thelivu
Nandukkal - Fossil crab
Aerva juce - Sirupeelai saru
Radish juce - Mullangi saru
The human dose prescribed traditionally by siddha practitioners for diseases like urinary obstructions, inflammation of urogenital tract, gravel in urine and bladder is 600 gm per day for an adult patient given daily in two fractions. ConConverting the human dose to an animal study dose, 10 mg of Nandukkal parpam was given to wistar rats daily in 1 ml water.
Experimental Animals
Eighteen male wistar rats weighing around 100-150 gm were on a standard pellet diet (Hindustan liver Ltd., India) and water given ad libitum. After acclimatisation in the test cages for a week they were divided into three groups, six in each group. Group I rats were controls, Group II rats were given 1 ml of ethylene glycol and 1 gm of ammonium chloride mixed in 100 ml water instead of plain drinking water to induce hyperoxaluria.[6,7] Group III rats were given the same quantum of ethylene glycol and ammonium chloride as in group II, in addition Group III animals were given Nandukkal parpam in the above mentioned dosage orally by gavage feeding. For all the three groups, food, water intake, weight and activity were noted two days once during the study period. The duration of the study was sixty days.
After noting the final body weight at the end of the study period i.e., 60 days two animals in each group were killed by cervical dislocation and the kidneys were removed for histopathological study. The evaluation is done by semi quantitative estimation of calcium oxalate crystals present in the section of kidney tissue of wistar rats as examined after H and E staining and microscope examination by a trained pathologist as well as by polarizing microscopy.
This project is cleared by the ethics committee constituted for KJ Hospital, Research and Post Graduate Centre and KJ Research Foundation, Chennai - 600 084. KJ Research Foundation is a registered Institution with CPCSEA (Committee for the purpose of control and supervision of experiments on animals) for ‘Experiment and Research on animals’.
RESULTS AND DISCUSSION
Ethylene glycol, a hyperoxaluria inducing lithogen has been found to be a reliable inducer of oxalate lithiasis in rats.[8] Ethylene glycol is converted to endogenous oxalic acid by the liver and ammonium chloride an agent that decreases urinary pH is supposed to upset the enzyme sorting mechanism in the tubular cells in the kidney.[7,8]
The H and E stained kidney sections were examined using plain and polarised light. The number of crystals in kidney sections of group II and III rats were counted in a semi quantitative manner under light microscope with a magnification factor of 240 (6 x 40) Group II animals receiving ethylene glycol and ammonium chloride showed an average of 230 crystals in 50 high power fields whereas group III rats receiving the above along with drug showed only an average of 10 crystals in the 50 high power fields (p < 0.001) (Table 1). Fig. 1a and 1b shows the H and E stained sections of kidney of Group II and III rats under light microscopic examination. Further H and E stained kidney sections of Group II rats when observed under plain and polarised light, kidney sections showed numerous microcalculi including distortion of tubule in some. Group III animals on nandukkal parpam administration showed very few crystals. Under polarizing microscope calcium oxalate crystals appeared bright white and birefriengent. Fig. 2a and 2b shows the H and E section of kidney of Group II and III rats under polarizing microscopic examination. Further the presence of crystals in Group II was also confirmed by application of von Kassa’s stain (Fig. 3), which converts the birefringent material into black precipitates.[9]
TABLE 1
Mean and SD values of crystals observed by light microscopy of H and E section of the kidneys of Group II and III wistar ratsGroup II Group III Statistical Significance No. of Fields Observed No. of crystals seen per field(Mean ± SD) No. of Fields Observed No. of crystals seen per field(Mean ± SD) 50 4.36 ± 6.91 50 0.22 ± 0.54 p<0.001
CONCLUSION
Ethylene glycol induced calcium oxalate crystals in the kidney of wistar rats in this study was significantly reduced by the administration of the siddha herbal combination drug Nandukkal parpam. ‘Nandukkal Parpam’ is used by siddha practitioners for management of urolithiasis in human beings. Controlled scientific studies in patients can be of immense use to establish the efficacy of this traditional Indian medication.
ACKNOWLEDGEMENT
Authors express their gratitude for their advise and support received from Dr. Sare Paul, Senior Consultant Pathologist, KJ Hospital, Chennai, Dr. B Murali Manohar, Prof. and Head Department of Pathology, Madras Veterinary College, Chennai, Dr. VR Seshadri, Secretary, IMCOPS and Dr. B Loganathan, Department of Biophysics, CLRI, Chennai.
REFERENCES
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3.Preminger GM. Is there a need for medical evaluation and treatment of nephrolithiasis in the ‘Age of lithotripsy"? Seminars in Urology 1994; 12 : 51-64.
4.Saeed R Khan. Calcium phosphate/calcium oxalate crystal association in urinary stones : implications for heterogeneous nucleation of calcium oxalate. J Urol 1997; 157 : 376-83.
5.Formulary of siddha medicines. The Indian Medical Practitioners Co-operation Pharmacy and Stores Ltd., Chennai. 1993; 225.
6.Lyon ES, Borden TA, Vermeulen CW. Experimental oxalate lithiasis produced with ethylene glycol. Invest Urol 1966; 4 : 143-51.
7.De Bruijn WC, Boeve ER, Van Run PRWA, Van Miert PPMC, Romijn JC, Verkoelen CF, Cao LC, Schroder FH. Etiology of experimental calcium oxalate monohydrate nephrolithiasis in rats. Scanning microscopy 1994; 8 : 541-50.
8.Khan SR, Hackett RL. Calcium oxalate urolithiasis in the rat : is it a model for human stone disease? A review of recent literature. Scan Electr Microsc 1985; 11 : 759.
9.De Bruijn WC, Boeve ER, Van Run PRWA, Van Miert PPMC, de Water R, Romijn JC, Verkoelen CF, Cao LC, Schroder FH. Etiology of calcium oxalate nephrolithiasis in rats 1. Can this be a model for human stone formation? Scanning Microscopy 1995; 9 : 103-14.
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