Evaluation of Antiurolithiatic activity of Betula utilis in Rats Using Ethylene Glycol Model

 

Samir K. Shah*, Kruti M. Patel, Pinal M. Vaviya

Department of Pharmacology, Sardar Patel College of Pharmacy, Bakrol, Anand, Gujarat, India

 

ABSTRACT:

This study was aimed to evaluate the antiurolithiatic activity of alcoholic extract of Betula utilis in rats using ethylene glycol model. Standard drug used was Cystone. Healthy male Wistar rats weighing 180-200 g were selected. Ethylene glycol (0.75%) in drinking water was fed to all the groups (Groups II–VII) except normal control (Group I) for 28 days to induce urolithiasis for curative regimen (CR) and preventive regimen (PR). Groups III, IV served as CR and groups V, VI served as PR were treated with alcoholic extract of Betula utilis (250 and 500 mg/kg) Groups II and VII served as model control and standard (Cystone 750 mg/kg) respectively. Several parameters were used including urinary volume, urine pH, urine analysis, serum analysis, kidney homogenate and histopathology of kidney to assess the activity. The results indicated that the administration of alcoholic extract of Betula utilis to rats with ethylene glycol-induced lithiasis significantly reduced all the elevated biochemical parameters (calcium, phosphate, oxalate, creatinine, blood urea nitrogen and uric acid), restored the urine pH to normal and increased the urine volume significantly (p < 0.05) when compared to the model control. This study supports the usage of alcoholic extract of Betula utilis in urolithiasis.

 

 

INTRODUCTION:

Urinary stone disease has affected humankind since antiquity and can persist, with serious medical consequences, throughout the patient's lifetime. In addition, the incidence of kidney stones has been increased in western societies in the last five decades, in association with economic development. Most calculi in the urinary system arise from a common component of urine, calcium oxalate (CaO), representing up to 80% of analyzed stones^1,2,3. This may cause obstruction, hydronephrosis, infection, and hemorrhage in the urinary tract system. Surgical operation, lithotripsy, and local calculus disruption using high-power laser are widely used to remove the calculi⁴. Many remedies have been employed since ages to treat renal stones and most of them were from plants and proved to be useful^5,6,7.

 

The present day medical management of nephrolithiasis is either costly or not without side effects⁸. The current study was aimed to evaluate Betula utilis D. Don belongs to the family Betulaceae, commonly known as a Bhojpatra traditionally used as an astringent, expectorant, tridosha, convulsion and diarrhea⁹. It is also reported to use as an antimicrobial¹⁰, anti inflammatory¹¹, antioxidant and antihyperglycemic¹² and hepatoprotective action¹³. Bhojpatra used to dissolve stones in the kidneys¹⁴, the leaves of the plant show efficacy in treatment of urinary tract infections and in kidney and bladder stones¹⁵. Quercetin shows inhibitory effect on urinary crystal deposition¹⁶ while lupeol and    ß-sitosterole gives antiurolithic action in hyperoxaluria¹⁷. It has been reported that Crataeva tapia and Bergenia ligulata are the plant shows antiurolithiatic activity containing quercetin, lupeol and ß-sitosterol¹⁸. There is an evidence of constituent present in Betula utilis like Quercetin, Lupeol, ß-sitosterole, hyperoside, guaijavarim, Betulin and Betulic acid so on the basis of above finding we indented evaluation of the antiurolithiatic activity Betula utilis in rats using ethylene glycol model.

 

MATERIALS AND METHODS:

Procurement of Plant:

Betula utilis leaves were collected from the Himalaya natural Nursery, Dehradun.

 

Authentication of Plant:

The Plant was authenticated by Medicinal Plants Survey and Collection Unit, Government Agriculture University, Anand, India. Sample was preserved at Pharmacognosy department of Sardar Patel College of Pharmacy (SPCP/ Herbarium/122440903001).

 

Preparation of Alcoholic Extract¹⁹:

The leaves were dried at room temperature until they were free from moisture, then leaves were subjected to size reduction to get a coarse power then passed through sieve no.40 to get a uniform power. Then around 500g of power was subjected for Soxhlet extraction with 90% ethanol along with use of benzene as pre-extractive solvent. After extraction solvent was distilled off. Extract was concentrated on water bath to dry residue. (Percentage of yield was 7.8% w/w)

 

Preliminary Phytochemical Investigation²⁰:

The extract was used for qualitative determination of phytoconstituents like tannins (phenolic compound), steroids, triterpenoids, flavonoids, alkaloids, carbohydrates and proteins.

 

Experimental Animals:

Male rats of body weight range between 180-200 g were used for induction of urolithiasis. Rats were housed in polyethylene cages under a 12/12 h light/dark cycle at around 22°C and had free access to water and food. Experiment was conducted according to the CPCSEA guidelines and the study was approved by the Institutional Animal Ethics Committee (IAEC) (Registration no. SPCP/IAEC/RP-012/2013).

 

Induction of Urolithiasis²¹:

Ethylene glycol (0.75% v/v) in drinking water was fed to all groups except normal control for induction of renal calculi till the 28^th day.

 

Experimental design²²:

In this study, a total of 42 male rats were used. The rats were divided in seven groups of six rats each and studied for 28^th days. Ethylene glycol (0.75% v/v) in drinking water was fed to all groups except normal control for induction of renal calculi till the 28^th day. Group I served as normal control and received regular rat food and drinking water ad libitum. Ethylene glycol (0.75%) in drinking water was fed to Groups II–VII for induction of renal calculi for 28 days. Group III and IV received Alcoholic extract of Betula utilis (250 mg/kg, p.o.) treatment from 15^th day (Curative Regimen). Alcoholic extract of Betula utilis. (500 mg/kg, p.o.) Treatment from 15^th day (Curative Regimen). Group V and VI received Alcoholic extract of Betula utilis (250 mg/kg, p.o.) treatment from 1^st day (Protective Regimen). Alcoholic extract of Betula utilis. (500 mg/kg, p.o.) Treatment from 1^st day (Protective Regimen). Group VII received standard antiurolithiatic drug, Cystone (750 mg/kg, p.o.) from 15^th till 28^th day.

 

INVESTIGATION OF PARAMETERS:

Measurement of volume and pH of urine:

Urine samples (24 hr) were collected on 28^th day by keeping the animals in metabolic polypropylene cages. Animals had free access to drinking water during urine collection period. The volumes of urine from each group of animals were mea­sured²³. The acidity of the urine was tested using the pH meter²⁴.

 

Stone observation in fresh urine:

The rats were kept separately in metabolic cages and 24 hr urine samples were collected on the 28^th day. A drop of concentrated hydrochloric acid was added to the urine prior to storage at 4°C. The collected urine samples were centrifuged at 3000 rpm for 10 min. After centrifugation, the urine samples were examined under light microscope at 100 X lens to identify the presence of crystals²⁵.

 

Urine analysis:

Urine samples (24 hr) were collected on 28^th day by keeping the animals in metabolic polypropylene cages. Animals had free access to drinking water during urine collection period. Urine was used for the estimation of calcium and phosphate using commercially available kits (Span Diagnostics Ltd., India)and oxalate by colorimetric method²⁶.

 

Serum analysis:

On last day of experiment (Day 28^th), rats were anaesthetized using ether and blood was withdrawn by retro-orbital puncture using micro capillary tubes. Serum was separated by centrifugation at 7000 rpm for 15 min and used for estimation of creatinine, uric acid and urea nitrogen using commercially available kits (Span Diagnostics Ltd., India).

 

Kidney Homogenate Analysis:

The abdomen was cut open to remove both kidneys from each animal. Isolated kidneys are cleaned off extraneous tissue and preserved in 10% neutral formalin. The kidneys were dried at 80°C in hot air oven. A sample of 100 mg of the dried kidney was boiled in 10 ml of 1N hydrochloric acid for 30 min and homogenized. The homogenate was centrifuged at 2000 rpm for 10 min and the supernatant was separated and the calcium and phosphate contents in kidney homogenate were determined using commercially available kits (Span Diagnostics Ltd., India) and oxalate by colorimetric method²⁶.

 

Histopathology of Kidney:

To confirm the incidence of urolithiasis the animals were sacrificed and their kidneys were isolated and subjected to histopathological studies. The kidneys were cleaned off from extraneous tissue and transferred to 10% neutralized formalin solution (pH 7.4). Sections of kidney was fixed in paraffin, stained with hematoxylin and eosin and observed for histopathological studies²⁷.

 

Statistical analysis:

Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test using Graph Pad Prism Version 5.03. All values were expressed as Mean ± SEM and p<0.05 was considered as significant.

 

RESULTS:

Preliminary phytochemical investigations:

The alcoholic extract of Betula utilis was subjected to phytochemical investigation, the results revealed the presence of alkaloids, tannins, flavonoids, glycoside and carbohydrate in alcoholic extract of Betula utilis.

 

Measurement of Volume and pH of Urine:

There was significantly decrease urine volume as well as pH of urine in ethylene glycol treated group when compared with normal control (NC) group. However, treatment with Betula utilis extract in curative regimen (CR) (250 mg/kg and 500 mg/kg, p.o.) significantly increased urine output as well as pH of urine as compare to model control (MC) group. Also treatment with Betula utilis extract in preventive regimen (PR) (250 mg/kg and 500 mg/kg, p.o.) more significantly increased urine volume as well as pH of urine as compare to model control (MC) group. The animals receiving standard treatment Cystone (750 mg/kg, p.o.) showed significant increased urinary volume as well as pH of urine when compared to normal and calculi induced model control group (Table 1).

 

Stone observed in fresh urine:

The microscopic examination of urine of normal group of animal showed the absence of crystal or similar structure (Fig. A), while in case of calculi induced group, the urine sample showed abundant, large crystals of Calcium oxalate (Fig. B). In Curative Regimen both groups (CR-250 and CR-500 mg/kg, p.o.) showed better dissolution of the preformed crystal of Calcium oxalate (Fig. C, Fig. D). However, small fragments of crystals were seen in both the groups. In Preventive Regimen, both groups (PR-250 and PR-500 mg/kg, p.o.) effect clearly showed better prevention of stone formation along with the dissolution of stones (Fig. E, Fig. F). The Cystone (750 mg/kg, p.o.) treated animals showed very less or almost dissolved small crystals (Fig. G) as compared to model control group (Figure 1).

 

 

Table 1 Effect of alcoholic extract of Betula utilis on urine volume and pH of urine in ethylene glycol induced urolithiasis.

NC-Normal Control; MC-Model Control; CR-Curative Regimen; PR-Protective Regimen; STD-Standard. All values are expressed as Mean ± SEM for each group (n=6). One way ANOVA followed by Dunnett post-hoc test.^*p<0.05 Model control Vs Normal control, ^#p<0.05 Treatment group Vs Model control.

 

 

 

A	D
B	E
C	F
G

Figure 1 Representative microscopic images (100x) of fresh urine. A=NC, B=MC, C=CR 250, D=CR 500, E=PR 250, F=PR 500, G=STD 750.

 

 

Urine analysis:

In the present study, administration of 0.75% (v/v) ethylene glycol aqueous solution to male Wistar rats resulted in hyperoxalouria. Stone forming promoters like calcium, oxalate and phosphate excretion were grossly increased in calculi-induced model control animals. Both curative and preventive regimen groups at (250 and 500 mg/kg, p.o.) by Betula utilis treatment and Cystone (750 mg/kg, p.o.) were significant (*p<0.05) lowered the elevated levels of these stone forming promoters in urine (Group III-VI and VII) as compared to calculi-induced model control group (Table 2).

 

 

 

Table 2 Effect of alcoholic extract of Betula utilis on calcium, phosphate and oxalate ofurine in ethylene glycol induced urolithiasis.

NC-Normal Control; MC-Model Control; CR-Curative Regimen; PR-Protective Regimen; STD-Standard. All values are expressed as Mean ± SEM for each group (n=6). One way ANOVA followed by Dunnett post-hoc test.^*p<0.05 Model control Vs Normal control, ^#p<0.05 Treatment group Vs Model control.

 

Serum Analysis:

Serum analysis showed that creatinine, uric acid and urea nitrogen levels were significant increased in 0.75 % (v/v) ethylene glycol treated model control group as compared to normal group. Both CR and PR at 250 and 500 mg/kg, p.o. by Betula utilis treatment and Cystone (750 mg/kg, p.o.) were significant (*p<0.05) lowered the elevated levels of serum creatinine, uric acid and urea nitrogen as compared to model control group (Table 3).

 

Table 3 Effect of alcoholic extract of Betula utilis on serum creatinine, urea and uricacid in ethylene glycol induced urolithiasis.

NC-Normal Control; MC-Model Control; CR-Curative Regimen; PR-Protective Regimen; STD-Standard. All values are expressed as Mean ± SEM for each group (n=6). One way ANOVA followed by Dunnett post-hoc test.^*p<0.05 Model control Vs Normal control, ^#p<0.05 Treatment group Vs Model control.

 

Kidney Homogenate Analysis:

The deposition of the crystalline components like calcium, oxalate and phosphate in the renal tissue (kidney homogenate) was increased in calculi-induced model control group. Both CR and PR at 250 and 500 mg/kg, p. o. by Betula utilis treatment and Cystone (750 mg/kg, p. o.) were significant (*p<0.05) reduced the kidney homogenate contents of these stone forming constituents (Group III-VI and VII) as compared to calculi-induced model control group (Table 4).

 

Table 4 Effect of alcoholic extract of Betula utilis on calcium, phosphate and Oxalate in kidney homogenate in ethylene glycol induced urolithiasis.

NC-Normal Control; MC-Model Control; CR-Curative Regimen; PR-Protective Regimen; STD-Standard. All values are expressed as Mean ± SEM for each group (n=6). One way ANOVA followed by Dunnett post-hoc test.^*p<0.05 Model control Vs Normal control, ^#p<0.05 Treatment group Vs Model control.

 

 

Figure 2 Representative microscopic images (100 xs) of histopathology of kidney. A=NC, B=MC, C=CR 250, D=CR 500, E=PR 250, F=PR 500, G=STD 750.

Kidney Histopathology:

Histopathological examination of kidney revealed the normal glomeruli and tubular region with the absence of calcium oxalate (CaOX) crystals in normal control rats. While in the model control group, severe glomeruli damage, numerous and large size CaOX crystal deposition in the renal tubule and dilation of the proximal tubules with intestinal inflammation were observed in the renal tissue. Treatment with Cystone and alcoholic extract of Betula ulitis decreased glomeruli damage, fewer numbers and smaller size CaOX crystal deposition in a dose dependent manner in comparison to the calculi-induced group.

 

DISCUSSION:

Urinary super saturation with respect to stone forming constituents is generally considered to be one of the causative factors in calculogenesis. Evidence in previous studies indicated that after 14 days period of ethylene glycol (0.75% v/v) administration, renal calculi was formed in the young male albino rat composed mainly of calcium oxalate²⁸. Stone formation in ethylene glycol-fed rats is caused by hyperoxaluria, which cause increased renal retention and excessive excretion of oxalate in urine⁸. In this study, oxalate and calcium excretion were increased in calculi-induced animals (Group II). An increase in urinary phosphate is also observed in calculi induced rats (Group II). Increased urinary phosphate excretion along with oxalate stress seems to provide an environment appropriate for stone formation by forming calcium phosphate crystals, which induce calcium oxalate deposition. Treatment with the Betula ulitis restored the phosphate level, thus reducing the risk of stone formation²⁹. In urolithiasis, the glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in the urinary system. Due to this, the waste products, particularly nitrogenous substances such as urea, creatinine and uric acid get accumulated in blood. Increased lipid peroxidation and decreased levels of antioxidant potential have been reported in the kidneys of rats supplemented with a calculi producing diet. Oxalate has been reported to induce lipid peroxidation and to cause renal tissue damage by reacting with polyunsaturated fatty acids in the cell membrane. In calculi-induced rats (Group II), marked renal damage was seen by the elevated serum levels of creatinine and uric acid and blood urea nitrogen³⁰. Histopathological examination of kidney sections derived from ethylene glycol induced urolithic rats showed dilation of the proximal tubules along with interstitial inflammation that might be attributed to increased oxalate and calcium level in kidney³¹. In present study treatment with alcoholic extract of Betula utilis decreased the number and size of calcium oxalate deposits in different parts of the renal tubules and also prevented damages to the tubules and calyxes. These may be due to the anti-inflammation action, which is further supported by the evidence that natural flavonoids have been reported to exhibit anti-inflammatory and analgesic activities³². However, the curative treatment with product Betula ulitis and cystone caused diuresis and hastened the process of dissolving the preformed stones and prevention of new stone formation in the urinary system. The diuretic effect of Betula ulitis was evident from urine volumes collected when compared to the model control group.

 

REFERENCES:

1.       Ravindra VK, Navneet BG, Alagawadi KR and Rudraprabhu VS. Effect of Moringa oleifera Lam. root – wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol. 105; 2006: 306–4.

2.       Vyas BA, Vyas RB, Joshi SV and Santani DD. Antiurolithiatic activity of whole-plant hydroalcoholic extract of Pergularia daemia in rats. J Young Pharm. 3; 2011:36–40.

3.       Tania AV, Cristina DD, Ana PS, Maria TR, Antonio JL and Caden S. Evaluation of the antiurolithiatic activity of the extract of Costus spiralis Roscoe in rats. J Ethnopharmacol. 66; 1999: 193–8.

4.       Hadjzadeh MA, Khoei A, Hadjzadeh Z and Parizady M. Ethanolic extract of Nigella sativa L seeds on ethylene glycol-induced kidney calculi in rats. Urol J. 4; 2007:86–90.

5.       Purnima A, Basavaraj CK and Vishwanathswamy AH. Antiurolithiatic and antioxidant activity of Mimusops elengi on ethylene glycol-induced urolithiasis in rats. Indian J Pharmacol. 42; 2010:380–3.

6.       Vargas SR, Perez GR, Perez GS, Zavala SM and Perez GC. Antiurolithiatic activity of Raphanus sativus aqueous extract on rats. J Ethnopharmacol. 68; 1999:335–8.

7.       Hossein H, Ali-Reza K, Zahra K and Vahideh M. Antiurolithiatic Activity of Pinus Eldarica Medw. Fruits Aqueous Extract in Rats. Urol J. 7; 2010:232–7.

8.       Bahuguna Y, Rawat MM, Juyal V and Gupta V. Antilithiatic effect of flower Jasmin auriculatum Vahl. Int J Green Pharm. 3; 2009:155–63.

9.       The Ayrvedic Pharmacopoeia of India. Bhurjah. 5: 21.

10.     Kumaraswamy MV and Kavitha HU. Antibacterial activity of Betula utilis against some pathogenic bacteria. Advances in Biological Research. 2008: 21-25.

11.     Kumaraswamy MV and Satish S. Free radical scavenging activity and lipoxygenase inhibition of WoodfordiafructicosaKurz and Betula utilis.African Journal ofBiotechnology.7 (12); 2008: 2013-2016.

12.     Ahmad R, Shrivastav S and Maurya R. Antihyperglycemic activity of Betula utilis. Indian J. Sci. Technol. 1(5); 2008.

13.     Duraiswamy A, Ravat M and Gupta S. Hepato protective Activity of Betula utilis bark on D-galactosamineindused hepatic insult. World Journal of Pharmacy and Pharmaceutical Science. 1; 2012: 456-471.

14.     Khare CP. Indian Meditional Plant. Betula utilis D.Don. Springer. 2007; pp. 91-92.

15.     Singh S, Yadav S, Sharma P and Thapliyal A. Betula utilis, a Potential Herbal Medicine. International Journal Pharmaceutical and Biological Archives. 3(3); 2012: 493-498.

16.     Park HK, Jeong BC, Sung MK and Park MY. Reduction of oxidative stress in cultured renal tubule cells and preventive effect on renal stone formation by the bioflavonoid quercetin. The Journal of Urology. 179(4); 2008: 1620 -1626.

17.     Varatharana S, Coothan K and Palanithan V. Antiurolithic effect of lupeol. Journal of Natural Product. 71(9); 2008: 1509-1510.

18.     Sharma P, Patil D and Patil A. Nephrolytic action of Cratia tapia. International Journal of Pharmaceutical Science and Research. 4(2); 2013:582-589

19.     Kotiyal JP and Shridhar N. Isolation and cheractrization of chemical constituent of leaves of Betula utilis. 2008: 1-6.

20.     Kokate CK, Purohit AP and Gokhale SB. Pharmacognosy. Nirali Prakashan, Pune.1990.

21.     Pawar AT, Gaikwad GD, Metkari KS, Tijore KA and Ghodasara JV. Effect of Terminalia chebula fruits extract on ethylene glycol induced urolithiasis in rats. 2; 2012: 99-103.

22.     Purnima A, Koti B and Vishwahathswamy A. Anti Urolithiatic activity of Mimusups elengi Extract On Ethylene Glycol Induced Urolithiasis In. Rats. Ind. J. of Pharmaco. 42(6); 2010: 380-383.

23.     Dixit P, koti BC and Vishwanathswamy AHM. Antiurolithiatic activity of Crashcal on ethylene glycol induced urolithiasis in rats. J Pharm Sci.4; 2014: 30.

24.     Baheti DG and Kadam SS. Antiurolithiatic activity of some traditional medicinal plants against calcium oxalate induced urolithiasis in rats. International Journal of Pharmaceutical, Chemical and Biological Sciences.3; 2013: 1276.

25.     Dharmalingam SR, Madhappan R, Chidambaram K, Ramamurthy S, Gopal K, Swetha P and Senthil KL. Anti-urolithiatic activity of Melia azedarach Linn leaf extract in ethylene glycol-induced urolithiasis in male albino rats. Tropical Journal of Pharmaceutical Research. 13; 2014: 391.

26.     Hodgkinson A. Determination of oxalic acid in biological material. Clin. Chem. 16; 1970: 547.

27.     Chinnala HM, Shanigarm S and Elsani MM. Antiurolithiatic activity of the plant extracts of Solanum virginianum on ethylene glycol induced urolithiasis in rats. International Journal of Pharmacy and Biological Sciences. 3; 2013: 328.

28.     Anbu J, Suman S, Swaroop Kumar SL, Satheeshkumar R, Nithya S and Kannadhasan R. Antiurolithiatic Activity of Ethyl Acetate Root Extract of Ichnocarpus frutescens using Ethylene Glycol Induced Method in Rats. J Pharm Sci Res. 3; 2011: 1182–9.

29.     Atmani F, Slimani Y, Minouni M and Hacht B. Prophylaxis of calcium stone by Hibiscus sabdariffa on experimentally induced nephrolithiasis in rats. BJU Int. 92; 2003:137–43.

30.     Atef M and Attar A. Antilithiatic influence of spirulina on ethylene glycol-induced nephrolithiasis in male rats. Am J Biochem Biotechnol. 6; 2010:25–6.

31.     Gilhotra U and Christina A. Effect of Rotula aquatica Lour. on ethylene-glycol induced urolithiasis in rats. Int. J. Drug Dev. and Res. 3 (1); 2011: 273-280.

32.     Chavada K, Fadadu N, Patel V, Patel G and Gandhi R. Effect of flavonoid rich fraction of Citrus medical linn. on ethylene glycol induced urolithiasis in rats. Journal of Drug Delivery and Therapeutics. 2(4); 2012: 109-116.

 

 

 

Received on 24.03.2017       Accepted on 25.05.2017     

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