Antinephrolithiatic Potential of Macrotyloma uniflorum Aqueous Extract in Rats

 

 

VDN Srinivas*, G Sumalatha, PPV Jagannath, D Siva Kishore, P Madhu Kiran and M Jhansi Rani

Department of Pharmacology, Hindu College of Pharmacy, Amaravathi Road, Guntur, Andhra pradesh

 

 

ABSTRACT:

Urolithiasis is a major health problem with its high morbidity, high cost of management and potential for end-stage renal disease. The aim of our study is to cure and prevent the recurrent stone formation. Herbal drugs claim many promising remedies in urolithiasis. An attempt is made in this direction in the current work by using Macrotyloma uniflorum. The experimental model selected for the present study is subcutaneous administration of gentamicin (40mg/kg/day) for 8 days and dietary supplementation with 5% ammonium oxalate starting at day 5 of the treatment regimen to induce calcium oxalate urolithiasis. On administration of aqueous extract of Macrotyloma uniflorum in preventive and curative groups, moderate to few crystals are observed along the mild appearance of edema and dilation in tubules and crystals are present focally indicating the ability of aqueous extract of Macrotyloma uniflorum to dissolve the pre-formed stones. The parameters monitored in the present study are Kidney weight, calcium and oxalate deposition in kidney and the histological studies support the calcium and oxalate deposition data in kidney. Thus aqueous extract of Macrotyloma uniflorum showed significant antinephrolithiatic activity.

 

KEYWORDS: Urolithiasis, Macrotyloma uniflorum, calcium oxalate stones.

 

 

INTRODUCTION:

The incidence of urolithiasis is high in developing countries like and the northern and north-western regions of India. These regions can be termed an endemic stone-forming belt, due to a dietary pattern rich in cereals and pulses. Urolithiasis is a consequence of complex physio-chemical processes and the major contributory factors are urinary super saturation, crystallization, calculogenesis and matrix formation. The sequence of events in the formation of any urinary stone can be: urinary saturation super saturation, nucleation crystal growth crystal aggregation crystal retention stone formation

 

Urolithiasis is the third most common disease of the urinary tract and it is affecting the human beings since the earliest days. The recognition of different types of urinary calculi also resulted in more varieties of medical treatment. Still recurrence rates continue to be high with one of every two patients having another stone with in 5 years1. So, there is a need to develop new drugs to prevent the recurrence of kidney stones. . In modern era, herbals are seen as potential medicine for a variety of diseases often viewed to supercede the pharmacological efficacy of allopathic drugs.

 

In most cases of the urolithiasis, the common component is either calcium oxalate (75-90%) or magnesium ammonium phosphate to an extent of 5-10%2, 3­. In Indian indigenous system of medicine Macrotyloma uniflorum (Fabaceae) is reported to be useful in the treatment of urinary stones. Although the plant is claimed to be useful in the treatment of urinary stones, there is no record of systematic pharmacological studies on the plant.


Figure-1

 

NORMAL

 

 

Figure-2: PREVENTIVE CONTROL

 

 

 

PREVENTIVE

Figure-3: CURATIVE CONTROL

 

CURATIVE

 

The present study was planned to assess the efficacy of the aqueous extract of seeds of Macrotyloma uniflorum as antinephrolithiac in albino rats. The animal model of nephrolithiasis involving tubular dysfunction or injury by administration of gentamicin parenterally and 5% ammonium oxalate mixed with standard rat pellet feed to induce calcium oxalate stones.

 

MATERIALS AND METHODS:

Source of Plant Seeds:

The plant seeds were collected from Basavaiah Ayurvedic Pharmacy, Chintaluru, Andhra Pradesh.

 

Preparation of aqueous extract:

Shade dried powdered seed material was mixed with water in 1:3 ratios for half an hour by heating over a water bath, kept overnight and filtered. The filtrate was concentrated (120mg/ml) in a china dish by heating over a water bath and was directly administered to animals.

 

PHARMACOLOGICAL STUDIES:

Experimental animals:

Male albino rats of Wistar strain weighing between 150-200gm were used. The animals were fed with commercial rat feed pellets (gold mohur) and were given water Ad libitum. They were kept in polypropylene cages in well-ventilated room under hygienic conditions.

 

Acute toxicity studies:

Healthy adult albino rats were fasted overnight with free access to drinking water. They were divided into five groups each containing eight animals. Group-I animals were treated with distilled water (2ml/kg/p.o) and Group-II to Group-IV animals received 1, 2, 4 and 8gm/kg p.o of aqueous extract of Macrotyloma uniflorum respectively.

TABLE -1: Effect of Aqueous Extract of Macrotyloma Uniflorum on Kidney Weights

S. No

Group

Kidney weight gm/ 100gms of body weight

I

NORMAL

0.4± 0.013

II

PREVENTIVE-CONTROL

0.56±0.015*

III

PREVENTIVE

0.33±0.016**

IV

CURATIVE-CONTROL

0.5±0.016*

V

CURATIVE

0.39±0.0127**

*P<0.001 compared to normal

**P<0.001 compared to respective control groups

 

TABLE -2: Effect of Aqueous Extract of Macrotyloma Uniflorum on Deposition of Calcium and Oxalate in Kidney

S. No.

Group

Deposition in kidney weight mg / gm of weight tissue

 

 

Calcium

Oxalate

I

NORMAL

0.52±0.07

2.42±0.2

II

PREVENTIVE – CONTROL

2.5±0.3*

10.09±1.21*

III

PREVENTIVE

0.31±0.03**

5.65±0.36**

IV

CURATIVE-CONTROL

2.45±0.2*

13.4±0.63*

V

CURATIVE

0.96±0.1*

8.69±0.23**

*P<0.001 compared to normal

**P<0.001 compared to respective control groups

 

 

The animals were observed continuously for 2 hours, and then intermittently and at the end of 48 hours, the number of deaths was noted to calculate LD504.

 

Gross behavioral changes:

The animals were observed for behavioral, neurological and autonomic profiles during acute toxicity studies5

 

Antinephrolithiatic study- Calculi Producing Diet (CPD) MODEL:

To study the effect of aqueous extract of Macrotyloma uniflorum on various parameters of stone formation such as

1. Effect of extract on kidney weight. (Table 1)

2. Deposition of calcium and oxalate in kidney. (Table 2)

3. Histological studies of kidney. (Fig 1-3)

In the nephrolithiatic study gentamicin treatment predispose to stone formation by inducing some damage i.e. focal necrosis, loss of border membranes and the occurrence of membrane debris in the tubule lamina. So, membrane debris formed due to renal damage can act as nucleation sites for calcium oxalate crystallization. This pattern of calculi deposition in renal medulla region is similar to human urolithiasis. Gentamicin has been shown to inhibit calcium reabsorption in proximal tubule6. Kidneys of nephrolithiatic rats showed deposition of calcium oxalate crystals in renal calculi and at the papillary tips. Many were located sub epithelial often anchored to the basement membrane. Hyperoxaluria and calcium oxalate deposition was induced by gentamicin and calculi producing diet. The standard rat pellet feed was powdered and mixed with ammonium oxalate (5%) and again made into pellets. This feed is called as calculi producing diet (CPD) and it is fed to animals to induce hyperoxaluria and calcium oxalate calculi. The animals were divided into five groups of ten animals each and were put on treatment schedules as shown in Fig.4.

 

Administration of gentamicin (40mg/kg/s.c) for first eight days induced renal tubular damage and hemorrhages in the kidneys of experimental animals. Co-administration of calculi producing diet (5%ammonium oxalate with standard rat feed) caused an increase in the deposition of calcium and oxalate in the kidney and urinary excretion of calcium and oxalate in both the control groups when compared to normal animals.

 

Effect of Extract on Kidney weight:

A significant increase in kidney weight was noted in the preventive control and curative-control groups when compared to normal animals (p<0.001) (Table 1).

 

A significant decrease in kidney weight was observed in the preventive and curative groups treated with aqueous extract of Macrotyloma uniflorum when compared to their respective control groups (p<0.001).

 

Deposition of calcium and oxalate in kidney:

A significant increase (p<0.001) in the deposition of calcium and oxalate was observed in both preventive-control and curative-control groups when compared to normal animals.

 

Figure 4: Treatment Schedules For Antinephrolithiatic Study

 

After administration of aqueous extract of Macrotyloma uniflorum for 15 days in the preventive group, a significant (p<0.001) decrease in calcium and oxalate deposits were observed when compared to the preventive-control group. These results indicate the efficacy of aqueous extract of Macrotyloma uniflorum in preventing the calcium oxalate type of stone formation in the kidney.

 

In the curative group, administration of aqueous extract of Macrotyloma uniflorum for 15 days caused a significant decrease (p<0.001) in calcium and oxalate deposition when compared to the curative-control group. This evident the significant effect of the aqueous extract of Macrotyloma uniflorum in dissolving the pre-formed calcium oxalate type of stones.

The calculi producing diet (CPD) has some advantages like:

·                      Rapid crystal and stone deposition with in 14 days.

·                      The pattern of stone deposition resembles that of human stone disease as majority of the crystals occur in the papillae.

·                      Renal function remains relatively normal.

 

Statistical Analysis:

The results are expressed as mean ± SEM and were statistically analyzed by Students t- test7.

 

RESULTS AND DISCUSSION:

Administration of aqueous extract of Macrotyloma uniflorum significantly reduced both calcium and oxalate deposition in the kidneys of both preventive and curative groups when compared to their respective controls. This indicates that aqueous extract of Macrotyloma uniflorum has beneficial effect in preventing calculi formation.

 

On histopathological examination both the preventive and curative control groups showed presence of calcium oxalate crystals in Lumina of tubules accompanied by edema and cast formation extensive intertubular hemorrhages and congestion of blood vessels. These histological observations support the presence and growth of renal calculi in renal medulla region as observed in human urolithiasis. On administration of aqueous extract of Macrotyloma uniflorum in preventive and curative groups moderate to few crystals are observed along the mild appearance of edema and dilation in tubules and crystals are present focally indicating the ability of aqueous extract of Macrotyloma uniflorum to dissolve the pre-formed stones. These histological studies support the calcium and oxalate deposition data in kidney. Thus aqueous extract of Macrotyloma uniflorum showed significant antinephrolithiatic activity.

 

CONCLUSION:

The present study has shown that formed urinary stones could be dissolved with aqueous extract Macrotyloma uniflorum and without the aid of surgical intervention. The recurrence of stones could also be prevented to a great extent. While it may not be possible straightway to altogether dispense with surgical and other invasive procedures, the use of Antinephrolithiatic drugs is definitely going to revolutionize the treatment of urinary stones.

 

ACKNOWLEDGEMENTS:

The authors are Thankful to Hindu College of Pharmacy for providing necessary laboratory facilities to carry out present research work.

 

REFERENCES:

1.     Bhairavi, C.R. Trivedi, L. Reber, E. Berta and L. Stong, New enzymatic method for serum uric acid at 500n.m, Clin chem. 24, 259 (1964).

2.     B.L. Dietrich, R. Blschlce and W. Schmandt, J. Urol. Nephrol., 24, 205 (1990).

3.     C. W. Vermeulen, W. J. Grove and R. Goetz, J. Urol. Nephrol., 64, 541 (1950).

4.     J. Crossland, Lewis Pharmacology, Churchill Living stone, Newyork, P.137 (1980).

5.     U. K. Sheth, N. K. Dadkar and G. K. Usha, Selected topics in experimental pharmacology Eds (1) P.124 (1972).

6.     Chahwala, S.B and Harpur. E.S. (1983): Gentamicin-induced hypercaciuria in the rat. Anta pharmacol Toxicol (53): 358.

7.     S. Bolton, Statistics in Remington’s pharmaceutical sciences, Gennaro, P.124 (1990).

 

 

 

Received on 07.07.2009

Accepted on 10.08.2009   

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(1): Jan.-Feb. 2010, 75-78