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Table of Contents
ORIGINAL ARTICLE
Year : 2019  |  Volume : 30  |  Issue : 3  |  Page : 99-106

Effect of hydroalcoholic extract of Cyperus rotundus L. Rhizome against ethylene glycol and ammonium chloride-induced urolithiasis in male sprague-dawley rats


1 Department of Ilmul Advia (Pharmacology), National Institute of Unani Medicine, Bengaluru, Karnataka, India
2 Department of Ilmul Advia (Pharmacology), Dr. MIJTU Medical College, Mumbai, Maharashtra, India

Date of Submission22-Nov-2018
Date of Decision30-Jan-2019
Date of Acceptance11-Feb-2019
Date of Web Publication20-Jun-2019

Correspondence Address:
Nasreen Jahan
Department of Ilmul Advia (Pharmacology), National Institute of Unani Medicine, Kottigepalaya, Magadi Main Road, Bengaluru - 560 091, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/UROS.UROS_136_18

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  Abstract 


Background: Cyperus rotundus L. is used in various dosage forms by Unani physicians in the treatment of urolithiasis. Aims and Objectives: The present study aims to evaluate the effect of hydroalcoholic extract of Cyperus rotundus in nephrolithiatic male Sprague Dawley rats. Materials and Methods: The animals were divided into 6 groups of 6 each. Group I received regular rat food and drinking water ad libitum. Group II to VI were treated with Ethylene glycol (0.75%, V/V) and Ammonium chloride (1%, W/V) in drinking water for 7 days to induce urolithiasis. Group II was sacrificed after 7 days administration of lithogenic agents however, from 8th day, group IV was treated with Cystone (750 mg/kg) and group V and VI with hydroalcoholic extract of Cyperus rotundus (100 mg/kg and 170 mg/kg, respectively) for further 14 days. Group III left untreated after 7 days administration of lithogenic agent till 14 days and sacrificed on 22nd day. Urine, biochemical parameters, kidney homogenate analysis and histopathology were carried out. Crystalluria analysed by light microscopy. Results: The test drug at both the doses showed significant reduction (P < 0.001) in number of urinary crystals. Test groups showed significant reduction in urine sodium (P < 0.05) and calcium (P < 0.001) while increased in urine magnesium. Serum creatinine (P < 0.01) and urea (P < 0.05) level significantly reduced in test groups. Histopathology of kidney showed almost normal kidney architecture. Kidney homogenate analysis showed significant reduction (P < 0.05) of calcium in group VI. Conclusion: The results showed that test extract has significant antilithiatic effect in terms of solute balance, reduction in crystal numbers and improvement in renal cell derangement.

Keywords: Antilithiatic activity, Cyperus rotundus, Unani medicine, urolithiasis


How to cite this article:
Jahan N, Bano H, Ahmed Makbul SA, Kumar B N, Mushir A. Effect of hydroalcoholic extract of Cyperus rotundus L. Rhizome against ethylene glycol and ammonium chloride-induced urolithiasis in male sprague-dawley rats. Urol Sci 2019;30:99-106

How to cite this URL:
Jahan N, Bano H, Ahmed Makbul SA, Kumar B N, Mushir A. Effect of hydroalcoholic extract of Cyperus rotundus L. Rhizome against ethylene glycol and ammonium chloride-induced urolithiasis in male sprague-dawley rats. Urol Sci [serial online] 2019 [cited 2019 Jul 16];30:99-106. Available from: http://www.e-urol-sci.com/text.asp?2019/30/3/99/260781




  Introduction Top


The worldwide increasing incidence of urolithiasis and its prevalence make it a matter of medical concern which usually starts with obstruction and if left untreated results in severe complications such as multiple infections and hemorrhage suggesting need of ideal medical care.[1] Risk factors for urolithiasis include age, sex, diet, geographic location, systemic and local medical conditions; genetic predisposition; and urinary composition. Urinary composition determines stone formation based on three factors: exceeding the formation product of stone-forming components, the quantity of inhibitors (e.g. citrate, glycosaminoglycan [GAG], etc.), and promoters (e.g. sodium, urates, etc.) in the urine. The anatomy of the upper and lower urinary tracts may also influence the likelihood of stone formation by predisposing to urinary tract infection or stasis.[2] The treatment of renal stone in modern medicine has not yet established and had number of side effects.

Renal stone and its treatments are recorded since ancient time. In Rhazes (854–932 AD) Al-Hawi, Avicenna's (980–1037 AD) Al-Qanoon, and Jurjani's (1041-1136 AD) Zakhir-e-khwarzam shahi, kidney and bladder calculi diseases have been described in detail.[3] Urolithiasis needs both preventive and curative therapy. Currently, there are no satisfactory drugs in modern medicine, which can dissolve the stone, and therefore, physicians remain to be depending on alternative systems of medicine for better relief.[4] Unani system of medicine uses all three natural sources of drugs, i.e. plant, animal, and mineral in different dosage forms.[5],[6] Nnumber of single and polyherbal or herbomineral formulations have been used for the treatment of urolithiasis by Unani physicians since ancient time. However, the scientific documentation of their use is not well established. Therefore, the need for exploration of natural sources has been assumed to be of great potential. Cyperus rotundus L. (Cyperaceae) is one of the most important drugs used frequently in Unani System of medicine for antilithiatic activity in various dosage forms, namely powder, paste, and confection. It is commonly known as “Nagarmotha,” found throughout India. It is a pestiferous perennial weed with dark green glabrous culms, arising from underground tubers.[7] In the Unani system of medicine, mainly, the rhizome of the plant is used as an antidote for scorpion sting, palpitation, indigestion, diarrhea and dysentery, jaundice, and ascites. The drug is also used as an antiemetic, vermifuge, lithotriptic, and diuretic.[8],[9],[10],[11] It is also an important ingredient of many polyherbal formulations such as Anqaroya Sagheer, Jawarish Jalinoos, and Dawae Bawaseer used in the above-mentioned ailments of the body.[12],[13] The major chemical constituents of C. rotundus rhizome are flavonoids, tannins, glycosides, alkaloids saponins, terpenoids, essential oils, carbohydrates, protein, and amino acids.[7],[14],[15],[16],[17] High-performance liquid chromatography analysis showed that total phenolic contents (gallic acid equivalents, mg/ml) of C. rotundus rhizome ethanolic extract (10 and 25 mg/ml) were 1.1758 and 2.0969 mg/ml, respectively, and purified flavonoid 10 and 25 mg/ml were 1.0159 and 2.0969 mg/ml, respectively.[15] The rhizome has been scientifically investigated and reported to possess analgesic,[18] antioxidant,[15],[17] anti-inflammatory,[19] antidiarrheal, antispasmodic,[20] antiplatelet,[21] antiallergic,[22] antimicrobial,[23] antiulcer,[24] antidysmenorrhea,[25] wound healing,[26] and activity against hyperlipidemia.[27] However, no scientific study was found for its antiurolithiatic effect; therefore, the present study was undertaken to evaluate its antiurolithiatic potential in male Sprague-Dawley (SD) rats.


  Materials and Methods Top


Animals

Male SD rats, weighing 200–250 g, were used for the study. The animals were procured from registered breeders. Before the experiment, the animals were allowed to get acclimatized for 1 week. They were maintained under standard laboratory conditions throughout the experimental period and provided standard rat diet and water ad libitum unless stated otherwise. They were housed in clean polypropylene cages at room temperature 25°C ± 2°C, humidity at 45%–55% with 12-h light and 12-h dark cycle. The animal care procedures and experimental protocol were in accord with the guidelines of Control and Supervision of Experiments on Animals. The present study was undertaken in the Department of Ilmul Advia (Pharmacology), National Institute of Unani Medicine (NIUM), Bengaluru. Before starting the experiment, the research protocol was submitted for ethical clearance. The Institutional Animal Ethics Committee of NIUM, Bengaluru, has approved the protocol vide its Ref. no. IAEC/IX/05/IA.

Chemicals and reagents

All chemicals and reagents used were of analytical grade. Ethylene glycol (EG) was procured from NICE chemicals Pvt. Ltd., Kerala. Ammonium chloride (AC) kit was obtained from CDH Pvt. Ltd. New Delhi. Reagents used for analysis of biochemical parameters were calcium (Arsenazo III method) and phosphorus was obtained from Pathozyme Diagnostic, EURO diagnostic systems Pvt. Ltd. Chennai, India. Magnesium kit was obtained from Coral Clinical Systems, Uttarakhand, India. Urea kit procured from Prism Diagnostics Pvt. Ltd. Thane, India. Creatinine was procured from AD, India, EURO diagnostic systems Pvt. Ltd.

Plant material and preparation of extract

The rhizomes of C. rotundus L. collected from the herbal garden of NIUM in the month of April were carefully washed and rinsed with tap water and shade dried at room temperature of 28°C ± 1°C for 15 days. The drug was identified by a Botanist and the voucher specimen was deposited in the herbarium of NIUM, Bengaluru (Ref. no. 15/IA/Res/2014). Dried rhizomes were pulverized in electric grinder to make a coarse powder. About 100 g of coarse powder was used for hot extraction using Soxhlet's apparatus in hydroalcoholic solvent (1:1 ratio) at the temperature of 80°C for 6 h. The liquid extract was cooled and filtered (Whatman filter paper no. 40). The filtrate was evaporated on water bath (80°C) till it dried completely. The yield percentage after extraction was found to be 12.79% w/w. The resultant brownish-black extract was stored in refrigerator for further use.[28],[29]

Dosage of the drug

The human therapeutic dose of C. rotundus L. is 7 g [8] as mentioned in the Unani classical literature. Acute and subacute toxicity studies were conducted [30],[31] and found that it is safe up to maximum dose of 2000 mg/kg b.w. The dose of the crude drug for SD rat was calculated by factor 7[32] and found to be 816 mg/kg. The yield percentage of extract with reference to the dose of crude drug was calculated as 12.79% w/w. Hence, the first dose of extract was found to be 104.8 mg/kg which has been rounded off as 100 mg/kg. To know the dose-dependent effect, a second dose was also calculated [33] and found to be 170.92 mg/kg, which was rounded off as 170 mg/kg. The extract was freshly prepared in 1 ml of 5% gum acacia suspension daily before each administration. The extract was administered orally by the gastric cannula.

Ethylene glycol and ammonium chloride-induced urolithiasis

This study was carried out by the method of Ahmed et al. and Kumar et al.,[28],[34] with a few modifications in the treatment schedule. Thirty-six male SD rats were used and divided into six groups of six animals each. Group I served as plain control and received regular rat food and drinking water ad libitum, while the animals of Groups II to VI were treated with EG 0.75% (V/V) and AC 1% (W/V) by adding in their drinking water for 7 days for induction of urolithiasis. All the animals from each group received regular rat food. The animals of Group II were sacrificed just after 7 days of administration of EG and AC and served as disease Control A, while the animals in Group III left untreated after 7 days administration of EG and AC till next 14 days and received regular rat food and water ad libitum and served as disease Control B thereafter they were sacrificed. From 8th day, the animals of Group I received 1 ml of 5% gum acacia, while the animals of Group IV was treated with cystone in the dose of 750 mg/kg [35] and served as standard control. The animals of V and VI groups were treated with the hydroalcoholic extract of C. rotundus L. in low and high dose (100 mg/kg and 170 mg/kg), respectively. The treatment was continued for further 14 days. At the end of treatment, all the animals of each group were sacrificed.

Urine analysis

On the 8th day and 22nd day of experiment, the animals of each group were placed singly in individual metabolic cage with water ad libitum for 24-h urine collection. The presence of crystals in urine was analyzed. About 10 μl of urine sample was transferred to slide and covered with cover slip. It was left for 30 min to get fix and dry. The number of calcium oxalate (CaOx) crystals were identified and counted using the light microscope (×40).[34],[36] For chemical analysis, 1 ml of urine sample was centrifuged at 2500 rpm for 5 min [37] and further observed for urinary chemical analysis. Urine was analyzed for the presence of calcium, phosphorus, creatinine, sodium, chloride, and magnesium by autoanalyzer.

Serum analysis

On the 22nd day, rats were anesthetized with thiopentone sodium (50 mg/kg IP). The abdomen was cut open and the blood samples were collected by cardiac puncture. Serum was separated by centrifugation at 10,000 rpm for 10 min.[38] Serum obtained were analyzed for serum calcium, creatinine, urea, and phosphorus by respective kits using autoanalyzer.

Kidney homogenate analysis

After collection of blood, both the kidneys were identified and carefully dissected out. One kidney from two animals of each group was sent for histopathological examination. While, the other kidneys of all the rats of each group were used for homogenate analysis. Isolated kidney was trimmed off from extraneous tissue then dried at 80°C in a hot air oven for 30 min. A sample of 100 mg of the dried kidney was boiled in 10 ml of 1 N hydrochloric acid for 30 min and homogenized using IKA T 10 basic Ultra-Turrax tissue homogenizer. The homogenate was centrifuged at 2000 ×g for 10 min [38] and the 6 ml supernatant were discarded. Remaining supernatant was used for the analysis of calcium and phosphorus content by autoanalyzer.

Statistical analysis

The data were analyzed using GraphPad software (Inc., San Diego, CA). ANOVA repeated measure with Tukey–Kramer multiple pair comparison test was applied for the parameters which passed normality test. P < 0.05 was considered as statistically significant.


  Results Top


Urine analysis

After 7-day administration of EG and AC, significant increase (P < 0.001) in number of CaOx crystals were found in all the groups when compared with plain control on the 8th day. On intragroup comparison at 22nd day, remarkable reduction (P < 0.001) in number of crystals were noted in all the groups when compared with the number of urinary crystals found on the 8th day. The same level of reduction (P < 0.001) was observed in both the test groups when compared with disease Control B on the 22nd day. The number of crystals in each test group after 14 days treatment with test drug was found almost equal to plain control [Table 1]. In disease control group, urine calcium and phosphorus increased while creatinine and magnesium level decreased significantly. On 22nd day after treatment with test drug, calcium and phosphorous decreased significantly (P < 0.05), while urine magnesium level increased significantly (P > 0.001), but no significant change was observed in chloride level in both the groups [Table 2].
Table 1: Effect of Cyperus rotundus L. on calcium oxalate crystals in male Sprague-Dawley rat urine

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Table 2: Effect of Cyperus rotundus L. on urinary parameters in male Sprague-Dawley rats

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Serum analysis

Serum calcium in disease Controls A and B (sacrificed after 7 days administration of EG and AC) was found to be increased (P < 0.01 and P < 0.05, respectively) when compared with plain control. Standard group showed significant reduction (P < 0.05) when compared with disease Control A. Test groups showed reduction in serum calcium level when compared with disease controls but statistically not significant. Serum creatinine in disease controls significantly increased (P < 0.01), whereas standard and test Groups A and B showed statistically significant reduction (P < 0.01) in comparison to disease controls. When serum urea of disease Controls A and B were compared with plain control, it was found to be significantly increased. Standard group and test Groups A and B showed significant reduction (P < 0.01 and P < 0.05, respectively) in comparison to disease Control B. Serum phosphorus was found to be increased in disease controls but not statistically significant. In test and standard groups, no significant reductions were observed when compared with the disease controls, but the values were found to be almost the same as in plain control [Table 3].
Table 3: Effect of Cyperus rotundus L. on serum parameters in male Sprague-Dawley rat

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Kidney homogenate analysis

The calcium level in kidney homogenate in disease Control A was found to be significantly increased (P < 0.05) when compared with plain control but not in disease Control B. In test Group B, significant reduction was observed (P < 0.05) when compared with disease Control A. The phosphorus level in disease Control A was found to be increased significantly (P < 0.05) when compared with plain control. In disease Control B, no significant difference was observed; clearly indicate the process of autohealing. In standard control, phosphorus level showed significant decrease (P < 0.05) when compared with disease Control A. In test Group B, phosphorus level was significantly decreased (P < 0.05) when compared with disease Control A [Table 4].
Table 4: Effect of Cyperus rotundus L. on kidney homogenate in male Sprague-Dawley rat

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Histopathological findings of the kidney

In histopathological study of the kidneys sections after hematoxylin and eosin staining, disease control groups showed cellular derangement, dilated and congested blood vessels, hypercellularity in Bowman's capsule, mononuclear inflammatory infiltration, and glomerular injury. On treatment with test extract, the histopathological changes reached almost normal level. The result clearly demonstrated the ability of test drug to heal the kidney injury and prevent the deposition of crystals [Figure 1].
Figure 1: Light microscopy (×400) of the kidney sections from plain control (a) showing intact architecture. In disease control (b), mild hypercellularity in glomerulus and in Disease control (c), mild hypercellularity, increased mesangial cells, congestion. In standard control (d), degeneration in tubular epithelial cells, dilated and congested vessels. In test Group A (e), mild congestion. In test Group B (f), eosinophilic material in tubules and normal blood vessels

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  Discussion Top


Supersaturation of urine is considered to be one of the important factors in stone formation. Studies show that 7–14 days administration of EG to male rats resulted in the formation of CaOx stones. Further renal CaOx deposition by EG and AC in rats frequently mimic to the stone formation in human.[39] In the present study, rats were treated with 0.75% EG and 1% AC for 7 days. It has been reported that EG being oxidized into oxalic acid by nonspecific dehydrogenase leads to hyperoxaluria which is considered to be the factor in the induction of lithiasis. EG metabolizes into CaOx monohydrate and produces renal mitochondrial toxicity similar to clinical CaOx renal calculi.[40]

Large number of CaOx crystals were identified in urine of male rats after administration of EG and AC.[39],[41],[42],[43] The present study revealed the similar findings as significant (P < 0.001) increase in CaOx crystals were found in each group on urinary microscopic analysis on the 8th day. However, after treatment with test, urinary crystals were reduced significantly (P < 0.001) in both the test groups when compared with disease controls. The result demonstrated that hydroalcoholic extract was highly significant in decreasing CaOx crystals in urine and thus very helpful in reducing crystal formation. The analysis of urine with respect to stone-forming agents provides a good indication of the risk of stones formation. In disease Control A and B groups, urinary calcium was increased significantly (P < 0.001) in comparison to plain control. It was also reported in previous studies that EG causes hypercalciuria, hyperphosphaturia, and hyperoxaluria. The increased urinary calcium is a factor favoring the nucleation and precipitation of CaOx from urine and subsequently crystal growth.[44] Hydroalcoholic extract of C. rotundus and cystone showed significant reduction (P < 0.001) when compared with disease Control B. Reduction in calcium level in urine provides less calcium to bind with oxalate and thus reduction in CaOx crystals which is evident in urinary microscopy observations. Reduction in urinary calcium also reduces supersaturation which is the main risk factor for stone formation.[45]

Urinary phosphorus in disease Control A and B was found to be increased as mentioned in previous studies.[41] Increased excretion of phosphorus has also been reported in stone formers and hyperoxaluric rats.[46] Excessive excretion of phosphorus in urine along with oxalate stress provides favorable environment for stone formation by forming calcium phosphate crystals, which epitaxially induces CaOx.[46] After 14 days treatment with test extract, the phosphorus excretion decreased but statistically not significant.

Urinary sodium was found to be significantly (P < 0.01) increased in disease Control A when compared with plain control, while in disease Control B, no significant elevation was observed. In test Groups A and B, sodium decreased significantly (P < 0.05), but highly significant reduction (P < 0.01) was noted in standard group. The result showed that the test drug is able in reducing the supersaturation by decreasing sodium in urine. In positive Controls A and B, urinary chloride was also found to be slightly increased after lithogenic treatment. On treatment with test and standard drugs to respective groups the chloride level reached upto normal. Excessive excretion of sodium and chloride in urine indicates diuretic activity. Some of the studies report increase in urine volume by lithogenic agents.[47] However, urine volume was found to be increased, but it was not statistically significant (data not shown), but excretion of sodium and chloride increased significantly in disease controls. The effect of test extract on sodium and chloride, clearly demonstrate that the test extract does not have any significant natriuretic and diuretic effect. Test drug has been reported for diuretic activity in the Unani literature.[8],[9] Hence, there is a need to investigate the test drug for diuretic activity using appropriate model. Urinary magnesium decreased significantly (P < 0.01) in disease Control A and B when compared with plain control. Magnesium (Mg) is an important inhibitor in stone formation. Magnesium binds with oxalate and becomes soluble, thus it decreases oxalate availability to bind with calcium and reduces the formation of CaOx and also decreases supersaturation.[42] Low level of magnesium is noted in stone formers as well as stone forming rats.[46],[48] On treatment with test drug, Mg level reached almost up to normal level. It was increased significantly in test Group A (P < 0.05) when compared with disease Control B. In test Group B, highly significant elevation (P < 0.001) was observed when compared with disease Control A. Although the disease Control B showed some elevation in magnesium level when left untreated for 14 days, it was not as much as in test groups. The results clearly indicate the efficacy of test drugs toward the elevation of magnesium level in urine and prevention of stone formation.

The supersaturation of urine with CaOx is an important factor in crystallization.[49] In the present study, a significant decrease in urinary calcium was observed along with the formation of CaOx stones. Urinary oxalic acid forms complexes with multiple cationic salts to form oxalate salts which are soluble when formed with magnesium, but when complex with calcium, forms insoluble CaOx thus causing crystalline precipitation of renal calculi of CaOx type.[48] Treatment with test extract significantly reduced CaOx excretion, thus reducing the supersaturation of urine. This might be responsible for preventing and also in dissolving the preformed CaOx type of stones by significantly decreasing calcium but increasing magnesium level in urine.

Serum creatinine and urea was found to be significantly increased in disease controls. Creatinine and urea are the markers of kidney and tubular damage. Glomerular filtration rate (GFR) is decreased due to injury in the kidney tissue. This decrease may be due to the obstruction to the flow of urine by the stones in the urinary systems. Due to this, the waste products, particularly nitrogenous substances increased.[50] Treatment with the test extract showed significant decrease in serum creatinine and urea. Urinary creatinine was found to be decreased in disease control groups. It may be due to decreased GFR which improved significantly (P < 0.05) in test Group A and B. The results clearly indicate that after treatment with test extract, inflammation and injury reduced as a result of which GFR improved. The test drug has been reported for antimicrobial and anti-inflammatory activities,[18],[19],[51] so the efficacy of the test drugs is justified by virtue of these properties. Serum calcium was found to be significantly increased in disease Control A (P < 0.01) and B (P < 0.05). Significant elevation (P < 0.05) was also observed in kidney tissue on homogenate analysis when compared with plain control. Subsequent to treatment with test drug serum calcium decreased significantly (P < 0.01) in test Group B, although the level of serum calcium decreased in test Group A statistically insignificant in comparison to disease Control A. The results of test extract were more significant than standard drug. With the above results, it can be inferred that test drug improved kidney tissue calcium disruption more in comparison to serum calcium.

In histopathological study of the kidneys of disease control groups A and B treated with lithogenic agents for seven days showed cellular derangement, dilated and congested blood vessels, hypercellularity in Bowman's capsule, mononuclear inflammatory infiltration with injured glomerulus. On treatment with test extract, the histopathological changes reached almost normal level. The findings demonstrated that the test drug in both the doses produced significant lithotriptic activity which is evident by the reduction of size of stone, rare presence of crystals in urine, and absence of crystal deposition in kidneys of treated groups. From the results, an inference can be drawn that the hydroalcoholic extract of the test drug are able to produce lithotriptic effect by breaking the crystals and prevent retention of crystals in kidneys.

Lithotriptic drugs contain several phytoconstituents and exert their effect through multiple mechanisms such as by increasing the urine volume, by inhibition of crystallization, by improving renal function, by improving the renal tissue antioxidant status, and by their antimicrobial, analgesic, and anti-inflammatory actions.[52] Several studies have shown that exposure to high levels of oxalate and CaOx crystals produce cellular injury mediated by membrane lipid peroxidation through intracellular reactive oxygen species generation. Therefore, a reduction in renal oxidative stress could be an effective therapeutic approach in the treatment of urolithiasis. In previous studies, the antioxidant effect of flavonoids in green tea [53] and Orthosiphon grandioxorum[48] decreased oxidative injury in renal tubular cells and CaOx deposition in rat kidney. The test drug contains flavonoids, saponin, and alkaloids [19] which exert their effect through antioxidant and smooth muscle relaxant effect to make expulsion of renal stone easy.[41] Stones are formed due to deficit in the crystallization inhibitory effect of urine and the presence of promoters when these conditions favor stone formation, the antiadherent layer of GAGs acts as a protective barrier against urinary stone. If this layer is damaged due to consequence of bacterial attack, a stone nucleus develops leading to full stone in the urinary tract. At this point, the drugs having antimicrobial property may be effective by protecting the antiadherent GAGs layer covering the epithelium of collecting system.[49],[54] The antimicrobial property of test drug has already been reported;[18],[51] therefore, it may be considered as one of the possible mechanism that the test drugs evolve as a part of its action.

On the basis of above results and discussion, it can be concluded that the test drug showed antiurolithiatic effect in dose-dependent manner it was comparable to the standard drug cystone. On intergroup comparison between test and standard groups, no significant difference was observed, thus it can be concluded that both the doses of test drug and standard drug are equally effective. Furthermore, the test drug possesses various pharmacological actions; therefore, it can be said that test extract produced effect by involving numerous actions.


  Conclusion Top


On the basis of results obtained, it can be concluded that test drug apart from having lithotriptic effect have also been described to possess antimicrobial, anti-inflammatory, and antioxidant activities. These effects may partially contribute in the process of lithotriptic effect. The Unani concept that litholytic drugs act by virtue of demulcent, resolvent, deobstruent, and lithotriptic properties are validated. However, further studies are needed in different animal models as well as clinical trials to prove the effectiveness of the test extract. Moreover, hydroalcoholic extract should also be standardized for its marker compounds responsible for antilithiatic effect.

Acknowledgment

The authors are extremely thankful to Prof. Abdul Wadud, HOD, Ilmul Advia, NIUM, Bengaluru, for providing necessary facilities for this work. The authors are also very grateful to Dr. Ghulamuddin Sofi, Reader, Ilmul Advia, for his valuable suggestions in statistical analysis.

Financial support and sponsorship

National Institute of Unani Medicine (NIUM).

Conflicts of interest

There are no conflicts of interest.



 
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