SHORT COMMUNICATION Effect of Cystone, a Herbal Formulation, on Glycolic Acid-induced Urolithiasis in Rats
S. K. Mitra,* S. Gopumadhavan, M. V. Venkataranganna and R. Sundaram
R & D Centre, The Himalaya Drug Co., Makali, Bangalore-562123, India
The effect of cystone, a herbal formulation, was studied on experimentally induced urolithiasis in rats. Oxalate urolithiasis was produced by the addition of 3% glycolic acid to the diet for a period of 42 days. Glycolic acid treatment resulted in a significant increase in the levels of calcium and oxalate in the kid- ney as well as in the total kidney weight. Also, the urinary levels of calcium, oxalate and inorganic phos- phorus were increased. Cystone treatment at 250, 500 and 750 mg/kg b.wt. p.o. for 42 days revealed a dose-related effect in the reduction of lithogenic substances, following glycolic acid induced urolithiasis. Simultaneous oral treatment with cystone at a dose of 500 and 750 mg/kg for 42 days, significantly re- versed the glycolic acid-induced urolithiasis, presumably by preventing the urinary supersaturation of lithogenic substances, especially of oxalate and calcium. The reduction of urinary and kidney oxalate levels by cystone may be due to its inhibitory action on oxalate synthesizing liver enzyme glycolate oxi- dase. These observations indicate that cystone can play an important role in the prevention of disorders associated with kidney stone formation. # 1998 John Wiley & Sons, Ltd. Phytother. Res. 12, 372–374 (1998)
INTRODUCTION cured from authentic sources and identified by Dr S.
Farooq, botanist of The Himalaya Drug Co. A voucher specimen was deposited in the herbarium of the R & D Urolithiasis is the third most common disorder of the Centre, Bangalore. The main constituents of this formu- urinary tract, the others being frequently occurring lation and its proportion are shown in Table 1. All plant urinary tract infections and benign prostatic hyperplasia powders were individually weighed and mixed. The drug (Hiatt and Friedman, 1982). The worldwide incidence of was administered as an aqueous oral suspension and the urolithiasis is quite high (Anderson et al., 1967) and in animals of the control group received water as vehicle. spite of tremendous advances in the field of medicine, The rats were divided into five groups of eight each. there is no truly satisfactory drug for the treatment of Rats of group I received the commercial diet and served renal calculi. Most patients still have to undergo surgery as control, group II was fed with a calculi-producing diet to be rid of this painful disease. Hyperoxaluria is the main (CPD: commercial diet mixed with 3% glycolic acid) for initiating factor for urolithiasis (Robertson and Peacock, 42 days (Chow et al., 1975). Groups III, IV and V re- 1980). Ayurveda, an indigenous system of Indian ceived 250, 500 and 750 mg/kg b.wt of cystone, respec- medicine, offers vast scope for the successful treatment tively once a day orally in addition to the CPD for the of urolithiasis. In the present study, cystone, a polyherbal same duration. formulation, mainly comprising plant drugs which are widely used for antilithic activity in traditional medicine was evaluated for its effects on experimentally induced Collection and analysis of urine samples. On day 42, urolithiasis in rats. immediately after administration of the respective assigned doses, the rats were housed in metabolic cages for 24 h urine collection. A drop of concentrated hydro- chloric acid was added to the collected urine and stored at MATERIALS AND METHODS 4° C. Levels of oxalate (Hodgkinson and Williams, 1972), calcium (Tsuyoshi Ohnishi, 1977) and inorganic Forty male rats of Wistar strain weighing between 180– phosphorus (Varley et al., 1980) were determined spec- 220 g were used in this study. The animals were acclima- trophotometrically. Sodium and potassium were esti- tized to standard laboratory conditions and maintained on mated using a flame photometer. 12 h light and dark cycle. The rats were fed with com- mercially available standard pelleted feed (Lipton India Assay of renal tissue samples. At the end of the Ltd., Bombay) and water ad libitum. experiment, on day 43, the rats were killed by cervical The constituent plants of the formulation were pro- dislocation and kidneys excised, washed with normal * Correspondence to: S. K. Mitra, R & D Centre, The Himalaya Drug Co., saline and weighed. The kidneys were dried at 80° C in a Makali, Bangalore-562123, India. hot air oven. A sample of 100 mg of the dried kidney was
CCC 0951–418X/98/050372–03 $17.50
# 1998 John Wiley & Sons, Ltd. Accepted 13 March 1998 CYSTONE ON UROLITHIASIS 373
Table 2. Effect of cystone on calculi-forming constituents in urine following 3% glycolic acid for 42 days Parameter Group I Group II Group III Group IV Group V Oxalate (mg/24 h) 12.30c 0.84 25.12 2.89 18.89 3.37 13.10b 2.95 12.92b 2.81 Calcium (mg/24 h) 4.26c 0.19 7.18 0.74 5.90 0.92 3.89b 0.36 3.87b 0.42 Inorganic phosphorus (mg/24 h) 0.923b 0.128 1.596 0.182 1.119 0.120 0.818b 0.136 0.800b 0.122 Sodium (mEq/24 h) 10.21c 0.94 4.23 0.79 6.58a 0.82 10.13c,d 1.12 10.48c,d 1.30 Potassium (mEq/24 h) 11.76c 0.91 6.49 0.69 8.46 1.54 10.81 1.98 10.02 2.14 Values are mean SE (n = 8). a p < 0.05, bp < 0.01 and cp < 0.001 compared with Group II.
Table 3. Effect of cystone on kidney weight and calculi-forming constituents in kidney following 3% glycolic acid for 42 days Parameter Group I Group II Group III Group IV Group V Wet weight (g/100 g b.wt) 0.340c 0.0054 0.423 0.0084 0.400b 0.019 0.358c 0.012 0.350c,e 0.009 Dry weight (g/100 g b.wt) 0.087c,d 0.0018 0.119 0.0015 0.112b 0.0020 0.096c,g 0.0022 0.094c,g 0.0016 Oxalate (mg/100 mg tissue) 0.449c,d 0.018 1.103 0.056 0.895a 0.061 0.563c,g 0.031 0.548c,f 0.039 Calcium (mg/100 mg tissue) 0.168c,d 0.0061 0.359 0.012 0.309a 0.016 0.216c,g 0.013 0.211c,f 0.020 Values are mean SEM (n = 8). a p < 0.05, b p < 0.02 and c p < 0.001 compared with Group II; d p < 0.01 Group I vs Group IV and V; e p < 0.05, f p < 0.01 and g p < 0.001 Group III vs Group IV and V.
boiled in 10 mL of 1 N hydrochloric acid for 30 min. The and Gershoff, 1965). Our results are in agreement with kidneys were then homogenized (Chow et al., 1974). The these studies, as shown by the significant increase in homogenate was centrifuged at 2000 rpm for 10 min, and kidney weight. The increase in urinary calcium and the supernatant separated. The estimation of oxalate and oxalate levels were also found to be highly significant. calcium was carried out by the method of Hodgkinson Cystone treatment at a dose of 250, 500 and 750 mg/kg and Williams (1972) and Tsuyoshi Ohnishi (1977), b.wt. revealed a dose related response. Cystone treatment respectively. at dose levels of 500 and 750 mg/kg b.wt showed a better protective effect. However, there was no significant Statistical analysis. The data of urinary and renal difference observed between 500 and 750 mg/kg b.wt of parameters were expressed as mean SEM. The results cystone treatment. These findings revealed that 500 mg/ were analysed statistically using ANOVA followed by kg b.wt of cystone is the minimum dose required for Dunnett’s t-test. The minimum level of significance was eliciting an optimal activity. Cystone treatment signifi- fixed at p < 0.05. cantly lowered the oxalate values (p < 0.01) probably by its inhibitory action on glycolate oxidase. Urinary sodium excretion was significantly elevated in the drug treated animals. Urinary potassium excretion was also elevated, RESULTS AND DISCUSSION though not significantly (Table 2). The reduction in the urinary oxalate level will be beneficial in preventing the Urinary supersaturation with respect to stone-forming urinary supersaturation with respect to oxalate. Calcium constituents is generally considered to be one of the and phosphorus play a vital role in renal calculogenesis. causative factors in calculogenesis. In this context, the Calcium and inorganic phosphorus levels were also changes in urinary oxalate levels are relatively much elevated in the rats receiving a calculi-producing diet. more important than those of calcium (Robertson and The increase in calcium excretion may be due to Peacock, 1980). In the present study, feeding 3% glycolic defective tubular reabsorption in the kidneys (Varalak- acid resulted in hyperoxaluria, which is known to be due shmi et al., 1990). Cystone treatment markedly reduced to the ready conversion of glycolic acid to oxalate by the the levels of calcium and phosphorus (p < 0.01) in urine oxalate synthesizing liver enzyme glycolate oxidase (Table 2). (Richardson and Tolbert, 1961). Hyperoxaluria is usually There was a significant increase in the kidney weight the initiating factor of oxalate urolithiasis. Glycolic acid, of animals receiving 3% glycolic acid which was almost the precursor of oxalic acid, is known to increase normalized in the cystone treated animals (Table 3). significantly the incidence of oxalate lithiasis (Runyan Glycolic acid feeding for 42 days resulted in renal tissue # 1998 John Wiley & Sons, Ltd. Phytother. Res. 12, 372–374 (1998) 374 S. K. MITRA ET AL.
deposition of calcium and oxalate. The increased depo- urine and renal tissue brought about by cystone treatment sition of calcium and oxalate in the renal tissue is known in calculosis is noteworthy. These effects could contri- to lead to papillary calcification and eventual calculi bute to the antilithic and lithotriptic property of this formation (Heutmann and Lehmann, 1980). A similar formulation. elevation in renal stone forming constituents in rats fed with CPD has been reported earlier (Baskar, et al., 1996.). Cystone administration significantly reduced both cal- cium and oxalate levels in kidneys, which is known to Acknowledgement prove beneficial in preventing calculi formation due to supersaturation of these lithogenic substances (Table 3). The authors express their thanks to Dr S. Farooq, botanist, The The reduction in the stone forming constituents in Himalaya Drug Co., for identification of different plant species.
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