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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 for 42 days.
Glycolic acid treatment resulted in a significant increase in the
levels of calcium and oxalate in the kidney as well as in the total
kidney weight. Also, the urinary levels of calcium, oxalate and inorganic
phosphorus were increased. Cystone treatment at 250, 500 and 750mg/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 reversed 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 oxidase.
These observations indicate that Cystone can play an important role
in the prevention of disorders associated with kidney stone formation.
Urolithiasis
is the third most common disorder of the urinary tract, the others
being frequently occurring urinary tract infections and benign prostatic
hyperplasia (Hiatt and Friedman, 1982). The worldwide incidence
of urolithiasis is quite high (Anderson et al., 1967) and in spite
of tremendous advances in the field of medicine, there is no truly
satisfactory drug for the treatment of renal calculi. Most patients
still have to undergo surgery to be rid of this painful disease.
Hyperoxaluria is the main initiating factor for urolithiasis (Robertson
and Peacock, 1980). Ayurveda, an indigenous system of Indian medicine,
offers vast scope for the successful treatment of urolithiasis.
In the present study, Cystone, a polyherbal formulation, mainly
comprising plant drugs, which are widely used for antilithic activity
in traditional medicine was evaluated for its effects on experimentally
induced urolithiasis in rats.
Forty male rats
of Wistar strain weighing between 180-220 g were used in this study.
The animals were acclimatized to standard laboratory conditions and
maintained on 12 h light and dark cycle. The rats were fed with commercially
available standard pelleted feed (Lipton India Ltd., Bombay) and water
ad libitum.
The constituent plants of the formulation were procured 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 Centre, Bangalore. The main constituents of this formulation
and its proportion are shown in Table 1. All plant powders were individually
weighed and mixed. The drug was administered as an aqueous oral suspension
and the animals of the control group received water as vehicle.
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Table 1: Main constituents
of Cystone
|
|
Plant Name
|
Family
|
Voucher specimen
|
Part used
|
Proportion (mg/g)
|
| Didymocarpus
pedicellata |
Gesneriaceae |
Fa-235
|
Flower |
290
|
| Saxifraga
ligulata |
Saxifragaceae |
Fa-236
|
Stem |
220
|
| Rubia
cordifolia |
Rubiaceae |
Fa-237
|
Stem |
72
|
| Cyperus
scariosus |
Cyperaceae |
Fa-238
|
Roots |
72
|
| Achyranthes
aspera |
Amaranthaceae |
Fa-239
|
Whole
plant |
72
|
| Onosma
bracteatum |
Boraginaceae |
Fa-240
|
Whole
plant |
72
|
| Vernonea
cinerea |
Compositae |
Fa-252
|
Whole
plant |
72
|
The rats were divided
into five groups of eight each. Rats of group I received the commercial
diet and served as control, group II was fed with a calculi-producing
diet (CPD: commercial diet mixed with 3% glycolic acid) for 42 days
(Chow et al., 1975). Groups III, IV and V received 250, 500
and 750mg/kg b. wt. of Cystone, respectively once a day orally
in addition to the CPD for the same duration.
Collection and analysis
of urine samples. On day 42, immediately after administration
of the respective assigned doses, the rats were housed in metabolic
cages for 24 h urine collection. A drop of concentrated hydrochloric
acid was added to the collected urine and stored at 4° C. Levels
of oxalate (Hodgkinson and Williams, 1972), calcium (Tsuyoshi Ohnishi,
1977) and inorganic phosphorus (Varley et al., 1980) were
determined spectrophotometrically. Sodium and potassium were estimated
using a flame photometer.
Assay of renal tissue
samples. At the end of the experiment, on day 43, the rats were
killed by cervical dislocation and kidneys excised, washed with
normal saline and weighed. The kidneys were dried at 80° C in a
hot air oven. A sample of 100mg of the dried kidney was broiled
in 10ml of 1 N hydrochloric acid for 30 min. The kidneys were then
homogenized (Chow et al., 1974). The homogenate was centrifuged
at 2000 rpm for 10 min, and the supernatant separated. The estimation
of oxalate and calcium was carried out by the method of Hodgkinson
and Williams (1972) and Tsuyoshi Ohnishi (1977), respectively.
Statistical analysis.The
data of urinary and renal parameters were expressed as mean ± SEM.
The results were analyzed statistically using ANOVA followed by
Dunnett’s t-test. The minimum level of significance was fixed
at p<0.05.
Urinary supersaturation
with respect to stone-forming constituents is generally considered
to be one of the causative factors in calculogenesis. In this context,
the changes in urinary oxalate levels are relatively much more important
than those of calcium (Robertson and Peacock, 1980). In the present
study, feeding 3% glycolic acid resulted in hyperoxaluria, which
is known to be due to the ready conversion of glycolic acid to oxalate
by the oxalate synthesizing liver enzyme glycolate oxidase (Richardson
and Tolbert, 1961). Hyperoxaluria is usually the initiating factor
of oxalate urolithiasis. Glyolic acid, the precursor of oxalic acid,
is known to increase significantly the incidence of oxalate lithiasis
(Runyan and Gershoff, 1965). Our results are in agreement with these
studies, as shown by the significant increase in kidney weight.
The increase in urinary calcium and oxalate levels were also found
to be highly significant. Cystone treatment at a dose of 250, 500
and 750mg/kg b. wt. revealed a dose related response. Cystone treatment
at dose levels of 500 and 750mg/kg b. wt showed a better protective
effect. However, there was no significant difference observed between
500 and 750mg/kg b. wt of Cystone treatment. These findings revealed
that 500mg/kg b. wt of Cystone is the minimum dose required for
eliciting an optimal activity. Cystone treatment significantly 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, though not significantly (Table 2). The reduction
in the urinary oxalate level will be beneficial in preventing the
urinary supersaturation with respect to oxalate. Calcium and phosphorus
play a vital role in renal calculogenesis. Calcium and inorganic
phosphorus levels were also elevated in the rats receiving a calculi-producing
diet. The increase in calcium excretion may be due to defective
tubular reabsorption in the kidneys (Varalakshmi et al.,
1990). Cystone treatment markedly reduced the levels of calcium
and phosphorus (p<0.01) in urine (Table 2).
|
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):
ap<0.05, bp<0.01 and
cp<0.001 compared with Group II. |
|
Table 3: Effect of Cystone
on kidny 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/100mg 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/100mg 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 ± SE (n=8):
ap<0.05, bp<0.02 and
cp<0.001 compared with Group II; dp<0.01
Group I vs Group IV and V;
ep<0.05, fp<0.01and
gp<0.001 Group III vs Group IV and V. |
There was a significant increase in the kidney weight
of animals receiving 3% glycolic acid which was almost normalized
in the Cystone treated animals (Table 3). Glycolic acid feeding
for 42 days resulted in renal tissue deposition of calcium and oxalate.
The increased deposition of calcium and oxalate in the renal tissue
is known to lead to papillary calcification and eventual calculi
formation (Heutmann and Lehmann, 1980). A similar elevation in renal
stone forming constituents in rats fed with CPD has been reported
earlier (Baskar et al., 1996). Cystone administration significantly
reduced both calcium and oxalate levels in kidneys, which is known
to prove beneficial in preventing calculi formation due to supersaturation
of these lithogenic substances (Table 3).
The reduction in the stone forming constituents in
urine and renal tissue brought about by Cystone treatment in calculosis
is noteworthy. These effects could contribute to the antilithic
and lithotriptic property of this formulation.
The authors express
their thanks to Dr. S. Farooq, Botanist, The Himalaya Drug Company,
for identification of different plant species.
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