Anti- Lithiatic Effect of Ipomoea batatas (L) Leaves and Tuberous Roots on Ethylene Glycol Induced Urolithiasis in Rats

 

R. Sathish*, G. Jeyabalan

Department of Pharmacy, Sunrise University, Alwar, Rajasthan, India.

 

ABSTRACT:

The present day medical management of lithiasis is either costly or not without side effects, hence the search for anti lithiatic drugs from natural sources has assumed greater importance. The anti-lithiatic activity of Ipomoea batatas leaves and tuberous roots at 200 and 400mg/kg doses in curative and preventive regimens were evaluated on ethylene glycol induced lithiasis in male wistar rats. Lithiasis was induced in rats by 0.75% ethylene glycolated water ad libitum for 28 days. On 28^th day urinary excretion of calcium, oxalate, phosphate, uric acid and protein were significantly (p<0.01) decreased and magnesium excretion was significantly (p<0.01) increased in both curative and preventive regimens of I.batatas leaves and tuberous roots when compared with lithiatic control animals. The significant reduction (p<0.01) of serum creatinine, uric acid, blood urea nitrogen levels and significant increase (p<0.01) in creatinine clearance were also produced by I.batatas leaves and roots in both curative and preventive regimens. The I. batatas leaves and roots supplementation also caused significant (p<0.01) diuresis in rats accompanied by increased diuretic index. The maximum responses were produced by ethanolic extract of I.batatas tuberous roots and the effects were significantly (p<0.01) more than that of ethanolic extract of I.batatas leaves in both preventive and curative regimens. These observations concluded that the I.batatas roots (sweet potato) can be used as a herbal remedy in the treatment of lithiasis.

 

 

 

 

INTRODUCTION:

The 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 and often severe pain in the abdomen, flank and groin¹. Urinary stone disease has afflicted humankind since antiquity and can persist, with serious medical consequences, throughout a patient’s lifetime. In addition, the incidence of kidney stones has been increased in western societies in association with economic development for the last five decades². The recurrence of urolithiasis is represent as a serious problem; patients who have formed a stone are more likely to form another, and thus stone prevention is highly recommended. Medical therapy can reduce the risk of stone recurrence in 80 to 90% of patients but may not successfully remove existing stones. So surgery should be reserved for stones which are larger, damages kidney tissue, severe urinary tract infections, blocks flow of urine etc. The options available for treatment are extracorporeal shockwave lithotripsy (ESWL), percutaneous nephrolithotomy (PCNL), ureteroscopic stone removal^3-6. The medical management of nephro-lithiasis is either costly or not without side effects hence the search from herbal sources has assumed most important for anti-lithiatic treatment. Many remedies have been employed from plants and proved to be useful to treat renal stones, though the rationale behind their use is not well established except for few plants⁷. India has been referred to as the medicinal garden of the world. The WHO has listed 20,000 medicinal plants in globally in which contribution of India is 15-20 %. Today around 50% of world population is totally depends upon the plant derived products as a primary health care with no side effects⁸. Ipomoea batatas (I.batatas) commonly known as sweet potato belongs to the family convolvulacae is the sixth most important food crop in the world. The sweet potato roots are useful in vitiated conditions of pitta, burning sensation, hyper dipsia, constipation, strangury, renal and vesicle calculi, diabetes, peptic ulcer, general weakness and used as refrigerant, laxative, aphrodisiac, diuretic and tonic^9,10. In India Sweet potato is using as folklore medicine for renal calculi, diabetes and general weakness by the village peoples of Thoppampatti in Tamil Nadu and traditionally given against renal calculi in West Bengal^11,12. The pharmacological effect of I. batatas on removal of kidney stone disease should be established to substantiate its traditional usage. While there are several animal models that are used to study hyperoxaluria and its consequences, the most commonly employed method to induce hyperoxaluria is to provide ethylene glycol (EG) in animal’s drinking water¹³. EG is readily absorbed along the intestine and is metabolized in the liver to oxalate. When ethylene glycol is metabolized by the body it produces four toxic metabolites, they are glycoldehyde, glycolate, glycolic acid and glycoxalate. These metabolite causes tissue destruction primarily from calcium oxalate crystal deposition.  Oxalic acid combines with calcium to form calcium oxalate crystals, which deposits in the kidney¹⁴. Hence in this study, an attempt has been made to establish the anti-urolithiatic property of I. batatas leaves and tuberous roots on ethylene glycol induced urolithiasis in male rats at both curative and preventive regimens along with its diuretic potential.

 

MATERIAL AND METHODS:

Plant Material and Extraction:

The fully matured leaves and fresh tuberous roots of I.batatas (L) lam were collected from the Vadipatti and Dindigal areas Tamil Nadu, India in the month of July 2015.  The plant were identified and authenticated by Dr.D.Stephen, Taxonomist, American College, Madurai. The dust free leaves and sliced tuberous roots were allowed to dry under shade in laboratory for 20 days and pulverized. About 1000g each leaves and tuberous root powders were macerated in ethanol individually in the flat bottom flasks and kept in room temperature for 7 days with occasional shaking. The extracts were filtered and solvents from extracts were evaporated by using rotary vacuum evaporator under reduced pressure of 300-500 mmHg at 50-60°C¹⁵.  The obtained semi-solid residues of ethanolic extracts of leaves and tuberous roots were henceforth called ethanolic extract of I.batatas leaves (EIBL) and ethanolic extract of I.batatas tuberous roots (EIBR). Both the leaves and tuberous root extracts of I.batatas were intended for preliminary phyto chemical evaluation¹⁶.

 

Experimental Animals

Male wistar rats weighing between 150-200gm were housed in polypropylene cages under proper humidity (50%), temperature conditions (252^oC) and maintained on normal 12-12 h day-night cycle. The animals were fed with commercial rat pellets (Amrut laboratory animal feed Ltd. Sangli, India) and were given water ad libitum. The experimental protocol and all the procedures were approved by Institutional animal ethical committee as per CPCSEA guidelines.

 

Determination of Anti urolithiatic activity

Animals were divided into eleven groups containing six animals in each group.  Lithiasis was induced by the administration of 0.75% ethylene glycol in water ad libitum to all groups viz., Lithiatic (Group II), standard (Group III) and test groups (Group IV to XI) except the normal control (Group I) for 28 days. The lithiatic group was not received any drug, standard group was received anti-lithiatic drug Cystone (750 mg/kg) from 15^th day onwards and test groups were maintained as prophylactic and curative regimens for both EIBL and EIBR. The groups IV and V were served as preventive regimens of I.batatas leaves (EIBL-P) and received EIBL 200 and 400 mg/kg respectively from 1^st day onwards to 28 days. The VI and VII groups were considered as curative regimens of I.batatas leaves (EIBL-C) and received EIBL 200 and 400 mg/kg respectively from 15^th day to 28^th day. The groups VIII and IX were served as preventive regimens of I.batatas roots (EIBR-P) and received EIBR at 200 and 400 mg/kg respectively from 1^st day to 28^th day. The groups X and XI were assigned as curative regimens of I.batatas roots (EIBR-C) and received EIBR at 200 and 400 mg/kg respectively from 15^th day to 28^th day. All the groups were received their respective drugs through oral route as suspension and were maintaining on commercial rat pellet diet throughout the experimental period^17-20.

 

 

Collection and Analysis of Urine and Serum             

On 14^th and 28^th day all animals were kept in individual metabolic cages specially designed to separate feces. The animals were free access of normal water but not food during urine collection and urine samples were collected for 24hrs in measuring cylinder. The collected urine samples were analyzed for calcium, magnesium, oxalate, phosphate, uric acid and protein. Calcium and magnesium were determined by colorimetric method, oxalate was determined by Hodgkinson and William’s method, phosphate, uric acid and protein in urine were determined using standard reagents kits. The water intake, urinary volume and Urine pH of all groups were also noted on 28^th day^21,22. After the experimental period (28^th day) animals were anaesthetized with diethyl ether, blood samples were collected from retro orbital in non heparinized tubes and centrifuged at 2000rpm for 20 min to obtain serum. The serum creatinine was determined by Jaffe’s kinetic method. Standard reagent kits were used to determine uric acid and blood urea nitrogen (BUN) and creatinine clearance was estimated by Cockcroft and Gault formula⁷.

 

Histopathology studies

On 28^th day animals were euthanized and abdomens were cut opened to isolate their kidneys for histopathological evaluation to confirm the incidence of lithiasis. The kidneys were washed, preserved rapidly in 10% neutralized formalin (pH7.4) and soaked in paraffin. The kidney tissues were cut at 5μm intervals and the slices were stained with hematoxylin and eosin. Tissue slices were photographed using optical microscopy under polarized light and pathological changes were observed ²³.

 

Determination of Diuretic activity

Male wistar rats weighing between 150-200gm were used. The method of Lipschitz et al. with minor modifications was employed for the assessment of diuretic activity. The animals were divided into six groups of six animals in each group. All the animals were fasted and deprived of food and water for 18 hours prior to experiment. All the rats were received 25ml/kg, p.o of 0.9% sodium chloride solution before drug administration to impose uniform salt and water load. The first group (control) received only 0.9% Nacl solution 25 ml/kg, p.o. The second group served as standard group and received Furosemide 20 mg/kg ,p.o. The groups III, IV has received EIBL at 200 mg/kg and 400 mg/kg and groups V and VI were treated with EIBR at 200 mg/kg and 400 mg/kg respectively suspended in 0.9% Nacl solution. After oral administration each animal were placed in an individual metabolic cages specially designed to separate feces and urine samples were collected at room temperature. The urinary volume (ml/24 hours) and the electrolytes Na⁺, K⁺, and Cl^- (mmol/l) excreted in urine were measured. The Na⁺, K⁺ concentrations were evaluated by Flame photometer and Cl^- concentration was estimated by titration with silver nitrate solution (N/50) using three drops of 5% potassium chromate as an indicator^24,25.

 

Statistical Analysis

The results were expressed as meanS.D and the difference among data were determined by using one way ANOVA followed by Dunnett’s test. As per suitability P<0.05 was considered as significant, P< 0.01 was considered as very significant.

 

RESULTS:

The yields of EIBL and EIBR were found to be 7.5% and 9.4% respectively. From preliminary phytochemical screening of I. batatas, it was found that carbohydrates, glycosides, phenols, tannins, proteins, amino acids, saponins, and flavonoids were present in both EIBL and EIBR. On 14^th day the treatment with I.batatas leaves and tuberous roots in prophylactic regimen (EIBL- P 200 mg/kg, EIBL- P 400 mg/kg, EIBR- P 200 mg/kg , EIBR- P 400 mg/kg)  were significantly (p<0.01) reduced the elevated  urinary calcium, oxalate, Phosphate, uric acid and protein when compared with lithiatic animals. Contradictorily the urinary excretion of magnesium was significantly (p<0.01) increased in the prophylactic treatment of both EIBL and EIBR (Table 1).

 

 

 

 

 

 

 

 

 

The concentrations of various ions in collected urine samples were investigated and found to fluctuate drastically after 28 days of treatment. On 28^th day urinary excretion of calcium, oxalate, phosphate, uric acid and protein were significantly (p<0.01) reduced, and magnesium were significantly (p<0.01) increased in all EIBL and EIBR treated groups when compared with lithiatic control group (Table 2). There is a significant (p<0.01) increase in urinary volume, pH and significant (p<0.01) decrease in water intake in prophylactic and curative treatments of EIBL and EIBR at both dosage levels when compared with lithiatic animals (Table 3). The maximum anti lithiatic responses were produced by EIBR- P 400 mg/kg treatment (Group IX).

 

 

Table 2: Effect of ethanolic extract of I.batatas leaves and roots in urinary excretion of ions on 28th  day
Groups	Treatment	Calcium	Magnesium	Oxalate	Phosphate	Uric acid	Protein
I	Normal control	4.92 ± 0.37	0.94 ± 0.08	0.34 ± 0.07	5.65 ± 0.42	4.55 ± 0.02	3.05 ± 0.12
II	Lithiatic control	9.84 ± 0.17#	0.51 ± 0.01#	3.82 ± 0.11#	10.54 ± 0.50#	15.02 ± 0.12#	6.84 ± 0.15#
III	Cystone 750 mg/kg	5.06 ± 0.10*	0.92 ± 0.06*	0.56 ± 0.23*	5.94 ± 0.26*	5.02 ± 0.32*	3.34 ± 0.15*
IV	EIBL- P 200 mg/kg	6.75 ± 0.97*	0.82 ± 0.04*	0.98 ± 0.21*	7.03 ± 0.21*	6.26 ± 0.16*	3.98 ± 0.91*
V	EIBL- P 400 mg/kg	6.61 ± 0.15*	0.85 ± 0.01*	0.76 ± 0.10*	6.22 ± 0.04*	5.92 ± 0.15*	3.81 ± 0.10*
VI	EIBL- C 200 mg/kg	7.51 ± 0.43*	0.75 ± 0.07*	1.83 ± 0.71*	7.23 ± 0.19*	7.30 ± 0.65*	4.21 ± 0.75*
VII	EIBL- C 400 mg/kg	7.03 ± 0.17*	0.78 ± 0.01*	1.57 ± 0.23*	7.18 ± 0.23*	7.24 ± 0.36*	4.14 ± 0.21*
VIII	EIBR- P 200 mg/kg	6.12  ± 0.63*	0.87 ± 0.04*	0.64 ± 0.20*	6.08 ± 0.08*	5.61 ± 0.14*	3.84 ± 0.17*
IX	EIBR- P 400 mg/kg	4.98 ± 0.15*$	0.95 ± 0.03*$	0.49 ± 0.03*$	5.86 ± 0.02*$	4.91 ± 0.14*$	3.19 ± 0.03*$
X	EIBR- C 200 mg/kg	6.95 ± 0.21*	0.83 ± 0.02*	1.28 ± 0.12*	6.43 ± 0.21*	6.12 ± 0.19*	4.01 ± 0.07*
XI	EIBR- C 400 mg/kg	5.92 ± 0.07*¥	0.90 ± 0.02*¥	0.58 ± 0.31*¥	5.98 ± 0.12*¥	5.10 ± 0.18*¥	3.71 ± 0.25*¥
Each value expressed as the mean ± SD (n = 6) one-way ANOVA; * - P<0.01 when compared with Lithiatic control group. #- P<0.01 when compared with Normal group, $ - P<0.01 when compared with group V, ¥ - P<0.01 when compared with group VII.

 

 

In the present study on 28^th day significant (p<0.01) increase in serum creatinine, uric acid, blood urea nitrogen levels and also significant reduction in creatinine clearance were indicating marked renal damage of lithiatic control animals. The standard Cystone, EIBL-P 200mg/kg, EIBL-P 400mg/kg, EIBL-C 200mg/kg, EIBL-C 400mg/kg, EIBR-P 200mg/kg, EIBR-P 400mg/kg, EIBR-C 200mg/kg, EIBR-C 400mg/kg were shown significant (p<0.01) anti-lithiatic activity as decrease in serum creatinine, uric acid, blood urea nitrogen levels and also increase in creatinine clearance when compared with lithiatic control animals. The Renal function was improved better by the EIBR-P 400mg/kg (Group IX) and it was observed as maximum anti-lithiatic activity out of all other groups (Table 4).

 

 

The histological structure of the ethylene glycol induced lithiatic kidney showed a lot of alterations when compared with the normal kidney. The lithiatic group also showed deposition of oxalate crystals in renal tubules and altered renal architecture. The high number of kidney tubules filled with colorless stone crystals and the crystals caused a further widening of tubules. The kidneys of EIBL and EIBR as prophylactic and curative treatments at both dosage levels showed lesser degeneration of epithelial lining, tubular dilatation and reduced renal damage (Figure1).

 

The diuretic indexes of urine samples were calculated to assess diuretic potential of I.batatas leaves and tuberous roots. The urinary volume of EIBL and EIBR (200 mg/kg and 400mg/kg) significantly increased (p<0.01) when compared with control group. The diuretic index of EIBL 200 and 400 mg/kg treatment were observed as 1.09 and 1.22 respectively. The diuretic indexes were found to be 1.28 and 1.34 in EIBR 200 and 400 mg/kg treatments respectively. The sodium, potassium and chloride excretions were significantly (p<0.01) increased by both EIBL and EIBR (200 and 400 mg/kg) when compared with control group.

 

Table 5: Diuretic effect of ethanolic extracts of I.batatas leaves and roots on experimental rats
Group	Urine volume (ml/24h)	Diuretic Index (T/C)	Na+ (mmol/l)	K+ (mmol/l)	Cl- (mmol/l)	Na+/k+	Saluretic index (T/C)
							Na+	K+	Cl-
I- Control	7.69±0.21	-	86.01±1.53	54.15±1.08	78.17±0.83	-	-	-	-
II - Standard	10.83±0.02*	1.41	169.96±1.20*	84.10±0.14*	97.20±0.44*	2.02	1.98	1.55	1.24
III – EIBL 200mg/kg	8.41±0.09*	1.09	122.09±1.41*	77.34±1.05*	83.87±0.12*	1.58	1.42	1.43	1.07
IV - EIBL 400mg/kg	9.42±0.43*	1.22	125.15±2.10*	80.72±0.62*	87.16±0.63*	1.55	1.46	1.49	1.12
V - EIBR 200mg/kg	9.83±0.01*	1.28	135.07±1.02*	82.95±0.25*	92.87±0.82*	1.63	1.57	1.53	1.19
VI - EIBR 400mg/kg	10.27±0.02*	1.34	158.25±0.94*	83.20±0.57*	95.16±0.56*	1.90	1.84	1.54	1.22
Each value expressed as the mean ± SD (n=6) one-way ANOVA, *-P<0.01 when compared with the control group

 

DISCUSSION:

In the present study Ethylene glycol induced renal calculi in male wistar rat model were used because according to previous studies, the oxalate metabolism, excretion and urinary system of Wistar rats are almost similar to human and renal crystal deposition is significantly less in female than male rats^26-29. Administration  of  ethylene  glycol  (0.75%,  v/v)  to  young  male  albino  rats  for 14 day  period forms renal calculi composed mainly of calcium oxalate. The biochemical mechanisms for  this  process  are  related  to  an  increase  in  the  urinary concentration  of  oxalate. Stone formation in ethylene glycol fed animals is caused by hyperoxaluria, which causes increased renal retention and excretion of oxalate³. The results of various urinary and blood parameters showed that lithiatic control rats has suffered from severe kidney damage by ethylene glycolated water, lithiasis was significantly (p<0.01) induced when compared with normal control rats. On 28^th day urinary excretion of calcium, oxalate, phosphate, uric acid, protein were reduced and magnesium were increased significantly (p<0.01) in all EIBL and EIBR treated groups when compared with lithiatic control group. In urolithiasis, the glomerular filtration rate (GFR) decreases due to the obstruction to the outflow of urine by stones in urinary system. Due to this, the waste products, particularly nitrogenous substances such as urea, creatinine, and uric acid get accumulated in blood³⁰. The urinary volume, pH were increased and water intake was decreased in all EIBL and EIBR treatments. The significant (p<0.01) anti-lithiatic activities of EIBL and EIBR were confirmed by decrease in serum uric acid, blood urea nitrogen, serum creatinine levels and also increase in creatinine clearance when compared with lithiatic control animals. The oxalate crystals are usually associated with injury of renal epithelial cells that facilitate the adhesion and tubular retention of crystals, as the injury to epithelial cells. The histological reports have shown sweet potato leaves and roots were restored normal cellular organization, renal architecture and also there was no crystal deposition and tubular dilatation. The cellular integrity was recovered and reduced renal damage in prophylactic and curative treatments of EIBL and EIBR at both dosage levels. The inter comparisons were made between EIBL and EIBR treatments in all observed parameters for further clarification of which part of sweet potato is most effective. From results of all parameters the EIBR-P 400mg/kg was significantly (p<0.01) more effective than EIBL-P 400mg/kg and the EIBR-C 400mg/kg was significantly (p<0.01) more active than EIBL-C 400mg/kg. The responses produced by EIBR as preventive and curative regimen at 200 and 400mg/kg dosage levels were more than that of EIBL (200 and 400mg/kg), this was shown its potential as an anti urolithiatic agent. Many of the herbals with anti-urolithiatic activity have also possessed significant diuretic activity. Diuretic activity, in turn, contributes to the anti urolithiatic action of these plants. Increased urine output increases the solubility of CaOx and other crystallizing salts and thus facilitates removal of small crystals and prevents their aggregation and growth^31,32. Diuretics are drugs that increase rate of urine flow and also increase the excretion rate of Na+ ion, which is a major determinant of extracellular fluid volume. The significant (p<0.01) increase in urinary volume, excreation of electrolytes were shown by EIBL and EIBR at both dosage levels. Usually Na+/K+ ratio greater than 10.0 indicate potassium sparing effect; but all the treatment groups in this study has lesser values so no potassium-sparing action was observed. Increased urine output by herbals may prove to be beneficial in kidney stones and for the treatment of mild urinary infections³³. The antilithiatic effect of I.batatas tuberous roots were comparably more than that of I.batatas leaves. Hence the I.batatas tuberous roots possessed more anti-lithiatic and diuretic potentials. The mechanism underlying anti-lithiatic activity of I.batatas tuberous roots are apparently related to increased diuretic and lowering the urinary concentrations of stone forming constituents. The phytoconstituents such as flavanoids, saponins, triterpenoids and alkaloids of many other plants has exhibited anti-urolithiatic  and diuretic activities by various mechanisms^34-36. So the phyto constituents like flavanoids and saponins of I.batatas tuberous roots may be responsible for anti-lithiatic and diuretic activities. These finding prompt the need for further studies to find exact constituent(s) related to anti lithiatic effect of I.batatas tuberous roots by which more effective treatment for lithiasis can be achieved.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 18.01.2018          Modified on 15.02.2018

Accepted on 20.03.2018       ©A&V Publications All right reserved