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RESEARCH ARTICLE
Year : 2009  |  Volume : 5  |  Issue : 19  |  Page : 83-89 Table of Contents     

Study the relative effect of spironolactone and different solvent extract of Tibulus terrestrison urolithiatic rats


1 East West College of Pharmacy, off Magadi road, Bangalore-27, India
2 Al-Ameen College of Pharmacy, Hosur road, Bangalore-27, India

Date of Submission04-Apr-2009
Date of Decision05-May-2009
Date of Acceptance15-May-2009
Date of Web Publication16-Feb-2010

Correspondence Address:
H Satish
East West College of Pharmacy, off Magadi road, Bangalore-27
India
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Source of Support: None, Conflict of Interest: None


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   Abstract 

Hyperoxaluria is the foremost initiating cause for urolithiasis in renal cavities, where urolithiasis is a common urinary tract disorder. There is much expectation of finding active anti-urolithiatic compounds from indigenous plant as there are still uses in therapeutic despite the progress in conventional chemistry and pharmacology in producing effecting drugs. Most patients still have to undergo surgery to get rid of painful disease. In our present study on anti-urolithiatic agents from plants which was compared with synthetic chemical substance. As we have selected the sequential extracts of Tribulus terrestris with pet ether, chloroform, alcohol and water and compared the activity with the synthetic steroidal drug spironolactone which was used on the lithiatic induced rats and estimated the effect of the extract by doing the invo and invitro analysis alongside by histopathological studies and found the ethanolic extract of the drug was found to be effective than other treatment.

Keywords: Tribulus terrestris , Ethylene glycol, Urolithiatic rats


How to cite this article:
Satish H, Raman D, Kshama D, Shivananda B G, Shridhar K A. Study the relative effect of spironolactone and different solvent extract of Tibulus terrestrison urolithiatic rats. Phcog Mag 2009;5, Suppl S2:83-9

How to cite this URL:
Satish H, Raman D, Kshama D, Shivananda B G, Shridhar K A. Study the relative effect of spironolactone and different solvent extract of Tibulus terrestrison urolithiatic rats. Phcog Mag [serial online] 2009 [cited 2019 Dec 10];5, Suppl S2:83-9. Available from: http://www.phcog.com/text.asp?2009/5/19/83/59789


   Introduction Top


Kidney stones are painful urinary disorders that start as salt/chemical crystals which precipitate out from urine. Under normal circumstances, the urine contains substances that prevent crystallization but for patients with this condition, these inhibitory substances are ineffective. Tiny crystals will pass out along with the urinary flow without causing problems. At least few of people will pass a kidney stone during their lifetime, producing some of the most severe pain possible, by increasing the stone concentration in the kindney. If the stone is large enough to block the tube (ureter) and stop the flow of urine from the kidney, it must be removed by surgery or other methods. It is also called Renal Calculus. Symptoms usually begin with intense waves of pain as a stone moves in the urinary tract. Typically, a person feels a sharp, cramping pain in the back and side in the area of the kidney or in the lower abdomen. Sometimes nausea and vomiting occur. Later, pain may spread to the groin. The pain may continue if the stone is too large to pass; blood may appear in the urine and there may be the need to urinate more often or a burning sensation during urination. If fever and chills accompany any of these symptoms, which may lead to infectioins.

The plant drugs survey of Tribulus terrestris narrates that, the drug has been utilized for pre-clinical and clinical activities. Tribulus terrestris L. is a member of the family Zygophyllaceae. It is an annual herb about 30-70 cm high and has pinnate leaves (of unequal length), yellow flowers and characteristic stellate shaped carpel fruits. It is widely distributed in Africa, Western Asia, China, Japan, Korea, Europe, Kuwait and India. The extracts from this plant have been used traditionally in treating a variety of diseases including hypertension and coronary heart disease, ocular inflammation and infertility in both sexes as an aphrodisiac and diuretics.

Spironolactone is a synthetic 17-lactone drug which is a renal competitive aldosterone antagonist in a class of pharmaceuticals called potassium-sparing diuretics, used primarily to treat heart failure, ascites in patients with liver disease, low-renin hypertension, hypokalemia, and Conn's syndrome as well as high blood pressure. On its own, spironolactone is only a weak diuretic, but it can be combined with other diuretics. As it is been used as a diuretic and most of the diuretic drugs have been used in urolithiasis treatment, so, it is been tried and compared with plant drug extract for the observation.


   Material and Methods Top


Preparation from the dried fruit

The collected drug Tribulus terrestris fruits was coarse powdered using mixer grinder, this coarse powder was subjected for sequential extraction process using soxhlet apparatus on using different solvent system like, Pet ether, chloroform, ethanol, and water, and there residual extracts were collected.

Animals

About forty two male, wistar strain weighing between 150-180gms were used in this study.

Methodology and treatment

The rats were divided in four groups of six in each; animals were acclimatized to standard laboratory conditions. The rats were fed with commercially available standard pelleted feed and water ad libitum [1],[3] . The sequential extracts of fruit Tribulus terrestris were administered as an aqueous oral suspension using tween 60 as suspending agent by PO route and control animals received water as vehicle. The rats groupings: Group-I Normal control + commercial feed, Group-II Calculi producing agent (CPA) 0.2% ethylene glycol aqueous for 20 days, [2] Group-III 0.2% ethylene glycol aqueous for 20 days +next 10 days 25mg/kgbody wt Spironolactone, Group-IV 0.2% ethylene glycol aqueous for 20 days +next 10 days 25mg/kgbody wt petether extract of Tribulus terrestris, Group-V 0.2% ethylene glycol aqueous for 20 days +next 10 days 25mg/kgbody wt chloroform extract of Tribulus terrestris, Group-VI 0.2% ethylene glycol aqueous for 20 days + next 10 days 25mg/kgbody wt alcohol extract of Tribulus terrestris, Group-VII 0.2% ethylene glycol aqueous for 20 days +next 10 days 25mg/kgbody wt water extract of Tribulus terrestris. All the groups were given commercial pellet diet for 30 days and water ad libitum.

Parameters investigation

Collection and analysis of urine samples


On day 31, immediately after administration of the respective assigned doses, the rats were housed in metabolic cages for 24hrs urine collection using metabolic cage and measured the urine volume and compared with the normal group. A drop of concentrated hydrochloric acid was added to the collected the urine and urine volume was measured and stored at 4°C. The Levels of Oxalate [4] , Calcium [5] , Inorganic phosphorous [6] , and Magnesium [7] were determined by autoanalyser / UV-spectrometer. Sodium [1] and Potassium [1] were estimated by Ion Selective Electrode (Rosh, name of the instrument).

Assay of renal tissue samples

At the end of the experiment, on day 33, the rats were sacrificed by cervical dislocation after anaesthetization, kidneys and liver were excised, washed with normal saline and kidneys were weighed, and one of the paired kidney was sent for histopathological studies. The other kidney was dried at 80°C in a hot air oven and dried weights were taken.

Kidney ion estimation

A sample of 100 mg of the dried kidney was boiled in 10 ml of 1N HCl for 30 min and homogenized. The homogenate was centrifuged at 5000 rpm for 15 min, and the supernatant separated. The estimation of oxalate [4] and calcium [5] was carried out

Liver enzyme estimation

The livers excised, were washed with cold 0.15 M KCI and homogenized and the supernatant were taken for Lactate dehydrogenase [8] and Glycolate oxidase [4] enzymes estimation.

Statistical analysis

The data of urinary, renal and enzymes parameters were expressed as mean ± SEM. The results were analyzed statistically using one way ANOVA. The minimum level of significance was fixed at P<0.05.


   Results and Discussions Top


The 0.2% ethylene glycol a caliculi producing agent (CPA) has successfully induced the oxalate urolithiasis in treated rats [9] . The synthetic drugs spironolactone, which were taken as standard drugs for the contrast with the different extracts of Tribulus terrestris. To read out the effects of the extracts and the others drugs, the Group comparison were done, that is Group-I normal compared with the others Groups II-VI and Group II compared with the Groups III- VI

Urine analysis:

Supersaturation of ions is the causative factors to cause urinary caliculi in the kidney and in the renal cavities, on orally inducing the 0.2% ethylene glycol for 20 days resulted in hyperoxaluria due to ready conversions glycolate to oxalate (refer group-II), which received 0.2% CPA. The percentage effect of ethylene glycol was seriously carried out by survival/toxicity studies on animals and found 0.2% as non-toxic and effective dose for the induction of calculi [9] .

Oxalate:

On comparision, the alcohol extract of Tribulus terrestris, has shown highly significant action to the spironalactone and water extract treated rats in reducing the urinary calculi, the reduction level of calculi in spironalactone was better than water extract and found significant, where the water extract was moderately significant when compared to normal control rats and CPA control rats, others pet ether extract and chloroform extract as not shown the reduction of oxalate ion concentration in urinary calculi. [Table 1]

Calcium and inorganic phosphate

The calcium and inorganic phosphate play vital role in renal calculogenesis, the increase in calcium, inorganic phosphate may be due to defection in tubular reabsorption in the kidney. The calcium, and inorganic phosphate levels were elevated in the rats receiving CPA. The rats receiving alcohol extract of Tribulus terrestris reduced more significantly on comparision to Group-I & II rats and Spironoloctone, has decreased the calcium, and inorganic phosphate concentrations significantly, and found effective, the water extract treated animals shown moderate reduction of ions and in the groups receiving pet ether extract, chloroform extract had increased the levels of calcium and inorganic phosphate levels.

[Table 1]

Magnesium ions

The magnesium ion content was found to be more in normal group when compared to control group. The groups treated with alcohol extract, water extract and spironoloctone has increased magnesium ion concentration and found reverse in the groups treated with pet ether extract, and chloroform extract.[Table 1]

Sodium and Potassium

There was increase in urine volume and shown diuretic effect in the groups treated with alcohol extract, water extract and spironaloctone, were the sodium and potassium ion concentration were found to be increased in the urine, except, in the group treated with spironalactone, the potassium ion concentration was reduced due to retention of potassium in the renal tubules, because, Spironalactone is found to be potassium sparing diuretic and increased out put of sodium, and the rat treated with pet ether and chloroform extract has decreased the urine volume which was in contrast to the output of sodium and potassium ion levels. [Table 1]

Kidney weight analysis

Due to the precipitation of ions the accumulation of ions in the kidney which leads to kidney weight variations on comparison to normal kidney, so, the wet and dry weight of the kidney were taken and compared between the groups, there was a significant variation were seen in both the wet and dry kidneys weight of animals receiving 0.2% glycolic acid daily of Group-II, which was almost decreased in kidney weight by the drug treated with alcohol and water extract of Tribulus terrestris, and the synthetic steroidal drug

Spironolactone on comparision to normal group and found increase in kidney weight in pet ether extract, and chloroform extract treated rats. [Table 2]

Kidney ion analysis

The glycolic acid induction for 20 days resulted in renal tissue damage and deposition of calcium oxalate. The increased deposition of calcium and oxalate in renal tissues is known to lead to papillary calcification and elevated caliculi formation. A similar elevation in renal stone forming constituents in rat fed with CPA (caliculi producing agent). The administration of alcohol and water extract of Tribulus terrestris, and the synthetic steroidal drug Spironolactone found more significant reduction in the calcium and oxalate levels and in the alcohol extract treated rat was more significant and other treated groups pet ether extract and chloroform extract did not reduce the calcium and oxalate ions. [Table 2]

Liver enzymes analysis

The liver is the major site of endogenous oxalate synthesis, where glycolate oxidaze and lactate dehydrogenase enzymes which will convert the glycolate to oxalate through glyoxalate, out of these enzymes the GAO is more actively effective in converting the glycolate to oxalate. The activity of the GAO enzyme was found to be less, in the rats treated with alcoholic and water extract of Tribulus terrestris, and the Spironolactone, the other treated groups Pet ether extract and Chloroform extract the activity of GAO enzyme increased in convertion of glycolate to oxalate. where LDH enzyme activity was reduced in the group treated with alcoholic extract and the other groups were ubiquitious distribution.

[Table 3]

Biopsy of Rat kidneys:

Histopathological studies reveal the condition of rat kidneys of normal, 0.2% ethylene glycol which is calculi producing agent (CPA) treated, the synthetic drugs and the different sequential extracts of Tribulus terrestris treated rats. The rat kidneys which were stained by Haemitoxylin and Eosin and were examined using microscope under 10X. The accumulation and dissolution of the calcium oxalate precipitates in the rats renal cavities (renal cortex and renal medulla) was recognised and identified in the section and compared with each other, the slides [Figure 3], [Figure 4], [Figure 5], [Figure 6] and [Figure 7],. which was compared to the slides of normal [Figure 1] and with (CPA) calculi producing agent [Figure 2] and respectively the scoring were done which is mentioned below the figures [4] .


   Conclusion Top


On detail study, the alcohol and water extracts of Tribulus terrestris, was found to be effective then Spironolactone in reducing stone forming constituents both in urine and renal tissues and also reduced, the enzyme activity of GAO and LDH.out of this the alcohol extract of Tribulus terrestris, were found to be more effective and highly significant in the reduction of calculi, which can be used as anti urolithiatic agent. The pet ether and chloroform treated groups were found be insignificant.

Acknowledgements[10]

 
   References Top

1.Mitra S.K., Gopumadhavan S., Venkataranganna M.V. Sunduram R. The effect of cystone, an herbal formulation, on glycolic acid-induced uro­lithiasis in rats. Phytotherapy Research. 12 : 1-3 (1998).  Back to cited text no. 1      
2.Yamaguchi satoshi H., Wiessner John T., Hasegawa Andrew Y., Hung Lin­da S., Mandel Gretchen S., Neil Mandel. Study of a rat model for calcium oxalate crystal formation without severe renal damage in selected condi­tions. International J. of Urology. 12 (s3): 290-298. (2005).  Back to cited text no. 2      
3.Varalakshmi P., Shamila Y. and Latha E. Effect of Crataeva nurvala in experimental urolithiasis. Journal of Ethnopharmacology. 28 : 313-321 (1990).  Back to cited text no. 3      
4.Hodgkinson A. and Willams A.N.N. An improved colorimetric procedure for urine oxalate. Clin. Chim. Acta. 36 : 127-132. (1972) Replace with newer ref 2000 onwards  Back to cited text no. 4      
5.Connerty H.V. and Briggs A.R. Estimation of calcium. Am. J. Cli. Pathol 45 : 290. (1966). Replace with newer ref 2000 onwards  Back to cited text no. 5      
6.Gomori. Estimation of Inorganic phosphate. J. Lab. Clin. Med. 27 : 955. (1942). Replace with newer ref 2000 onwards  Back to cited text no. 6      
7.Tietz N., Saunders W.B. Magnesium estimation. Text book of clinical chemistry. 974 (1986). Replace with newer ref 2000 onwards  Back to cited text no. 7      
8.King J. Lactate dehydogenase. Practical clinical enzymology. 191-208 (1965). Re­place with newer ref 2000 onwards  Back to cited text no. 8      
9.Cruzan George et al, Subchronic Toxicity of Ethylene Glycol in Wistar and F-344 rats. Related to Metabolism and Clearance of Metabolites Toxicological Sciences. 81 (2): 502-511 (2004).  Back to cited text no. 9      
10.Indian Herbal Pharmacopoeia. Topic Tribulus terrestris. 154-161 (1999).  Back to cited text no. 10      


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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