Pharmacological Screening of Ethanolic Extract of Plant Solanum XanthocarpumOn Nephrolithiasis Rats

About Authors:
Amar Patil*, Shraddha Kanase
M.Pharm (Pharmacology)    
Department Of Pharmacology, Tatyasaheb Kore College Of Pharmacy,
Warananagar. Dist;Kolhapur, India .
*appatil.tkcp@gmail.com

Abstract:
Solanum Xanthocarpumhas been known traditionally in Indian system of medicine for the treatment of various metabolic disease and disorders. Since, the present study was carried out to know its applicability in urolithiasis condition. In the present study, extracts of Solanum Xanthocarpumwas prepared with Ethanol and used for evaluation of antiurolithiatic activity. In this study we systematically evaluated its property by using Ethylene Glycol induced Albinorats. This study also involves state of the art animal model to elucidate its probable mechanism of industrial guidelines of FDA, USA for pre-clinical evaluation of anti-Nephrolithiasis drugs.

REFERENCE ID: PHARMATUTOR-ART-1468

Introduction :
Kidney stone disease is a multi-factorial disorder resulting from the combined influence of epidemiological, biochemical and genetic risk factors¹.
A kidney stone is a hard, crystalline mineral material formed within the kidney or urinary tract. Kidney stones are a common cause of blood in the urine(hematuria) and often severe pain in the abdomen, flank, or groin. Kidney stones are sometimes called renal calculi.

The condition of having kidney stones is termed nephrolithiasis. Having stones at any location in the urinary tract is referred to as urolithiasis, and the term ureterolithiasis is used to refer to stones located in the ureters.

Urolithiasis, referred to the formation of a stone in urinary tract, is one among the diseases that afflicted humans since antiquity. Despite tremendous advances accomplished in understanding the mechanisms governing the formation of such concretion, the disease remains an enigma since several factors intervene and interfere each other.

As many forms of mineralization, urolithogenesis encompasses several physico-chemical steps which occur either sequentially or concurrently that start with supersaturation, then nucleation, growth, and aggregation. Of course, based on their size, aggregated particles can be attached and retained within urinary tubule epithelium constituting an additional step in calculogenesis. According to clinical and epidemiological studies, calcium oxalate (CaOx) followed by calcium phosphate are the most frequently encountered crystalline components found in analyzed stones. Most stones do not contain one single crystal phase but rather a mixture of several different crystal phases. This leads to wonder which phase was initiated the crystallization process.The overall probability of forming stones differ in various parts of the world and is estimated as 1-5% in Asia, 5-9% in Europe, 13% in North America and the recurrence rate of renal stones about 75% in 20 years span. It occurs both in men and women but the risk is generally high in men and is becoming more common in young women¹.Solanum Xanthocarpumhas been known traditionally in Indian system of medicine for the treatment of various metabolic disease and disorders. Since, the present study was carried out to know its applicability in urolithiasis condition.

OBJECTIVES OF THE STUDY:
The present study was carried out with an objective to find out the effect of Ethanolic extract of Solanum Xanthocarpumon Ethylene Glycol induced Albinorats.

MATERIAL AND METHODS:
Eight male Wistar albino rats, weighing between 150 to 280g, were selected and allowed to acclimatize for a minimum ten days prior to the study. The rats were housed in room maintained of 21±1^o c, relative humidity 50-55% and 12hr light-dark cycle. The rats were caged with one animal in each polypropylene cage and were fed with standard food pellets and water ad libitum. throughout the study. All animal experiments and maintenance were carried out according to thecommittee for the purpose of control and supervision of experiments on animal (CPCSEA) guidelines. All the experimental procedures  were approved by the animal ethical commitee (IAEC).. After ten days the rats were divided into four groups, two rats in each group. 

CHEMICALS AND DRUGS:
In the present study, the root of Solanum Xanthocarpumwas collected in Warananagar.The plant was authenticated by Prof. S. Y. Jadhav, H.O.D. of Botany, Yashwantrao Chavan Mahavidyalay, Warananagar studies includes organoleptic tests, and macroscopic and microscopic observations were done in by, Prof. S. Y. Jadhav. Then voucher specimen is deposited at Science College and number is BSC /BOT/11/03. Soon after authentication, all leaves were dried at room temperature, until they were free from the moisture. Finally the root was subjected to get course powder and then passed through sieve no. 44 to get uniform powder. The sieve powder was stored in air tight, high-density polyethylene container before extraction. Ethylene glycol(Merck specialties private ltd, Mumbai.),ethanol (Merck specialties private ltd, Mumbai.).

Plant preparation:
The powdered root (500g) was subjected to hot continuous extraction (soxhlet) with ethanol. After the residue extraction, solvent was distilled off, and excess solvent was completely removed by using a rotary flash evaporator to get reddish-brown semi solid extract (yield: 28.17%). The obtained extracts were then evaluated for anti-urolithiasis activity.

Experimental design:
The rats were divided into 4 groups of 2 rats in each group.They were grouped as:
The ratsfrom all groups were administering the ethylene glycol except for rats from groupI, which served as Normal.
1)       Group I: Normal control will receive (saline) vehicle.
2)       Group II: Animal served as control.
3)        Group III: Animal was orally administered a standard (Spironolactone 2 mg/kg).
4)       Group IV: Animal were maintained on normal diet, along with oral administered of ethanol extract at 100 mg/kg, respectively.

Evaluation of antiurolithiatic activity:
The extract treatment was started the days of the administration of ethylene glycol and continued for 30 days, but the body weights of all animals were recorded at the beginning and at weekly intervals throughout the experiment. Twenty–four hour urine samples were collected using separator by placing each group in a metabolic cage during the last week. Urine samples were acidified with 2ml of 1M HCL and centrifuged at 100g for 10 min at 4^oc to remove contaminating Sediments aliquots were stored at 20^o c until they were assayed. After 30 days  treatment with ethanolic extract of solanum xanthocarpum, experimental blood samples from all the Groups were withdrawn by retro arbitral, blood  samples were allowed to clot at room temperature and the serum was separated by centrifugation at 1050g for 20 min. Serum samples were stored at 70^oc until analysis. The left and right kidneys were dissected out. The left and right kidneys were used for the measurement of dry and wet weight. The left and right kidneys were immediately fixed in 10% neutral buffered formalin for histopathological examination.

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Biochemical estimation:
1. Serum Analysis:Blood urea nitrogen, uric acid.
The test was carried out using diagnostic reagent kit (Span diagnostic Ltd. Surat, India) for the In vitro determination of Blood urea nitrogen, uric acid  analyzed by using an auto analyzer.

2. Serum Analysis:  Creatinine.
This method is based on the deproteinisation of samples with picric acid and the determination of creatinine in the supernatant by the addition of NaOH solution. The assay was performed exactly described in Arzneibuch (D. L.) DDR-’83. To 250 μ? of sample, 1.5ml of picric acid were added, mixed and centrifuged for 10 min at 3000g. The supernatant was mixed with 50 μ? of NaOH solution (final concentrations: 41 mmol/1 picric acid, 69 mmol/1 NaoH) and the absorbance was measured between the 20th and 30th min at 530 nm.

This method is based on the deproteinisation of samples with picric acid and the determination of creatinine in the supernatant by the addition of NaOH solution. The assay was performed exactly described in Arzneibuch (D. L.) DDR-’83. To 250 μ? of sample, 1.5ml of picric acid were added, mixed and centrifuged for 10 min at 3000g. The supernatant was mixed with 50 μ? of NaOH solution (final concentrations: 41 mmol/1 picric acid, 69 mmol/1 NaoH) and the absorbance was measured between the 20th and 30th min at 530 nm.

3. Urine analysis: Calcium oxalate crystals.
The test was carried out using diagnostic reagent kit (Span diagnostic Ltd. Surat, India) for the In vitro determination of calcium oxalate. The calcium present in the serum was precipitated with naphtyl hydroxamic acid (calcium reagent). The precipitated was then dissolved in EDTA reagent and calcium from this solution was complexed with color reagent to give a colored complex that was measured colorimetrically, and analyzed using an autoanalyzer.

4. Renal Tissue Samples analysis:
At the end of the experiment, on day 30, 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 100 mg of thedried kidney was broiled in 10 ml of 1 N hydrochloric acid for 30 min.

Results:
Effect of Solanum Xanthocarpum extract on Nephrolithiasis rats body weight changes:
Table 1, show significant changes in body weight in Ethylene Glycol induced rats comparable to Normal group rats. It was also noticed that significant changes in body weight in animals treated with Solanum Xanthocarpumethanolic (100 mg/kg p.o) compared to Standard drug in nephrolithiasis rats.

1 .Effect of Ethanolic Extract Solanum Xanthocarpumon body weight in Nephrolithiasis rats (0.2 ml/kg)

Table 1.

Effect of Solanum Xanthocarpumextract on body weight in nephrolithiasis rats was calculated from first day of study and every week regularly. The body weight of each animal was recorded one hour prior to administration of drug extract on recording day.

2. Effect of Ethanolic Extract Solanum Xanthocarpumon Urine sample  in Nephrolithiasis rats (0.2 ml/kg).
Calcium and oxalate excretion were significantly increases in 24h urine of ethylene glycol induced nephrolithiasis rats when compared with normal rats. It was also decreases significantly when treated with ethanolic extract of Solanum Xanthocarpumcompared with Group II and shows similar significance result with standard.

As per the report of urine analysis by GIRNAR Laboratory, Jaysingpur stated that as follows:

Table 2.

Effect of Solanum Xanthocarpumextract on nephrolithiasis rats was studied by urine biochemical analysis. Biochemical parameters were estimated by using Span diagnostic kits purchased from local market. Results obtained from serum biochemical analysis are expressed by presence of calcium oxalate crystals in test. Urine biochemical results were collected from each group and evaluated and considered as significant result.

3. Effect of Ethanolic Extract Solanum Xanthocarpumon Serum sample in Nephrolithiasis rats (0.2 ml/kg).
As per the report of serum sample analysis by GIRNAR Laboratory, Jaysingpur stated that as follows:

Table 3.  

Effect of Solanum Xanthocarpumextract on nephrolithiasis rats was studied by serum biochemical analysis. Biochemical parameters were estimated by using Span diagnostic kits purchased from local market. Result obtained from serum biochemical analysis are expressed as Mean ± SE and evaluated by student’s’ test. Serum biochemical results were collected from each group and statistically evaluated by Chi-square test for coming to conclusion. P value less than 0.05 was considered as significant. **p<0.01

4. Effect of Ethanolic Extract of Solanum Xanthocarpumon Dry and wet Kidney weight in Nephrolithiasis rats (0.2 ml/kg).

Table 4

Values are mean ± SE evaluated by student’s’ test. P value less than 0.05 was considered as significant. *p<0.01

Statistical analysis:
All the data collected in the present study are expressed as Mean ± SE and were analysed by students ‘t’ test for coming to conclusion.

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DISCUSSION:
The ethylene glycol-induced calcium oxalate urolithiasis model in rats has been characterized and extensively used in the study of the mechanism, therapy and prevention of stone formation.

In the past, since increased calcium intake was considered to be an important risk factor for stone formation, calcium restriction was recommended as an obvious intervention to prevent kidney stones in calcium stone-forming patients suffering from hypercalciuria. However, this practice has been questioned due to a large epidemiologic study which reported an increased risk for stone formation in subjects consuming a low calcium diet due to secondary hyperoxaluria, although these data had been largely indirect. Furthermore, several investigators have observed that calcium restriction has a deleterious effect on bone in hypercalciuric patients.

In healthy subjects, observed that an increased calcium intake can totally abolish the hyperoxaluria induced by an oxalate load. However, in an experimental model of genetic hypercalciuric rats, observed that, although urinary calcium proportionally increased with increasing dietary calcium content, a parallel decrease in urinary oxalate did not occur.

By knowing the biochemical studies Primary stone formation and recurrence of stone formation is one of the biggest challenges faced by urologists today and remain a major source of morbidity in humans. Despite intensive studies in the last decade many aspects of nephrolithiasis / urolithiasis, the complete pathogenesis and thus prevention, still remains to be clarified. Studies have concentrated on the metabolic defects in urine and mechanisms of supersaturation, reasons for cytotoxic damage of renal tubular cells, and interference with some natural inhibitors. It is believed that, like urinary metabolic defects, biochemical variations in blood are less likely to occur in first time stone formers than in patients with recurrent disease. Increased excretion of oxalate and deposition of calcium oxalate crystals in the renal tubules is associated with renal epithelial injury and that products of cellular damage can act as heterogeneous nucleators of both calcium oxalate and calcium phosphate crystals in animal model.

The body weights of rats at the beginning of the study were not significantly different between the four groups. However, at the end of the study, the rats treated with 0.75% Ethylene Glycol weighed less than those on the normal diet. The Group-III and Group IV treatment with Standard and Extract of Solanum Xanthocarpum significantly increases the level of body weight in urolithic rat.

The present study were urinary chemistry with respect to stone forming minerals will provide a good indication of risk of stone formation. In the present study, observed hypercalciuria in ethylene glycol induced urolithic rat might be a factor favoring the nucleation and precipitation of calcium oxalate of apatite (calcium phosphate) from urine and subsequent crystal growth. The Solanum Xanthocarpumsignificance reduced the level of calcium oxalate in urolithic rat.

Uric acid is known to promote calcium oxalate crystal growth. Predominance of the uric acid crystals in calcium oxalate stones and the observation that uric acid binding protein are capable of binding to calcium oxalate and modulate it crystallization also suggests its primary role in stone formation. In the present study, higher concentration of urinary uric acid was observed in ethylene glycol induced urolithiasis rats. Solanum Xanthocarpumtreatment restored the uric acid level to normal thus reducing the risk of stone formation.

The serum uric acid and BUN were remarkably increased in ethylene Glycol induced animal while serum creatinine was only slightly elevated in group-II indicating marked renal damage. However ethanolic extract of Solanum Xanthocarpum reatment in curative and prophylactic regimen significantly lowers the elevated serum level of creatinine, uric acid and BUN in Group IV.

There was a significant increase in the kidney weight of animals receiving 0.75% ethylene glycol which was almost normalized in the Solanum Xanthocarpumt reated animals (Table 4). ethylene glycol feeding for 30 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. Solanum Xanthocarpum 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. Solanum Xanthocarpum used traditionally in ayurveda, for various kind of the disease and disorders and an anti-tumor effect. Aphrodisiac effect, anthelmintic, diarrhea, anti-fertility effect, on the impact of blood vessels. The earlier photochemical analysis is demonstrated the presence of the alkaloids, Quinones, Glycosides, Saponin, Fat and oils, triterpenes and Steroids. It has also been evaluated antioxidant, anti-inflammatory, antimicrobial, anti-fungal, antistress, anti-diarrheal, and antiulcer properties. Recently, studies have implicated the generation of oxygen derived free radicals and lipid peroxidation as one of the most important mechanisms involved in the pathogenesis of Nephrolithiasis. Antioxidants are known to inhibit lipid peroxidation and scavenge free radicals. By knowing the above pathophysiology of nephrolithiasis  and the plant profile of  Solanum Xanthocarpumis having a essential chemical constituents that reduce the  formation of calculi in kidney as well as ureter and urinary bladder.The use of ethanolic extract of Solanum Xanthocarpum is also appeared to be safe for long-term usage, as there were no short term and long term toxicity reports. Therefore, it can be considered that use of Solanum Xanthocarpum extract will be beneficial, safe and effective in management of nephrolithiasis.

CONCLUSION:
On detail study, the alcohol extracts of Aristolochia indica,was found to be effective then Spironolactone in reducing stone forming constituents both in urine and renal tissues and also reduced, the calcium and oxalate out of this the alcohol extract of Aristolochia indica,were found to be more effective and highly significant in the reduction of calculi, which can be used as anti urolithiatic agent.  The result exhibited by ethanolic extracts of Solanum Xanthocarpumshownsignificant anti urolithiasis activity. Further studies will extensive investigation, isolation, purification of active phytoconstituetnts with potent antiurolithiatic activity.The presented data indicate that administration of ethanolic extract of Solanum Xanthocarpumto rats with ethylene glycol induced lithiasis reduced and prevented the growth of urinary stones, supporting folk information about the anti-urolithiatic activity of plant. Based on these results, we conclude that the Solanum Xanthocarpumethanolic extract is more potent in protecting the animals from nephrolithiasis. All of their effects observed in this study are similar to Spirinolactone treatment. Hence, this Solanum Xanthocarpum is considered as an anti nephrolithiasis drug in treatment of nephrolithiasis.

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