"Evaluation of Antiurolithiatic Potential of Moringa Oleifera Seed Extract"

Moringa oleifera Lam. (Sohanjna, Drumstick tree, Horseradish tree; family, Moringaceae) is a well-known plant, native to Pakistan, Bangladesh, India, Sri Lanka, Philippines and Africa. It is known as miracle tree as well as mother’s best friend. Almost every part of plant (i.e., leaves, seeds, bark, root and wood) is a rich source of vitamins, minerals (calcium, magnesium and phosphorus), carotenes and folic acid. Various parts of M. oleifera such as pods, roots and bark have been reported for kidney stone disease; however, no such data is available to show the antiurolithiatic potential of seeds. The aim of the study was to validate the antiurolithiatic potential of Moringa oleifera Lam. seeds against ethylene glycol-induced urolithiasis.


Introduction
Urolithiasis is a debilitating condition in which one or more calculi are formed in any part of the urinary system and the stones are classified according to their location in the urinary system, i.e., kidney stones, bladder stones, ureteral and urethral calculi. Kidney stones are classified on the basis of composition, i.e., calcium oxalate, calcium phosphate, uric acid, struvite and cystine stones.
The two main types of calcium oxalate (CaOx) crystals include calcium oxalate monohydrate and calcium oxalate dihydrate.
There are four main steps of stone formation, i.e., supersaturation, nucleation, aggregation and crystal growth. Supersaturation occurs when there is less amount of water and high concentration of solutes (calcium salts). Most of the renal stones are small and can easily pass out through the urinary tract without any irritation and pain. While some stones are larger and cannot pass through the urinary system and get attached to the renal epithelial cells causing damage through oxidative stress [1].
Urolithiasis is a common and serious problem throughout the world affecting approximately 12% of the population. In Asian countries, it is a recurrent life-threatening disorder and the recurrence rate is upto 50% during 5-10 years. In Pakistan, the prevalence rate of urolithiasis is about 16% [2]. For urolithiasis, men are at higher risk than women (3:1) which can be associated with many predisposing factors like levels of testosterone and nutritional differences. Diet containing higher amount of animal protein can enhance the risk of urolithiasis. High water intake can reduce the risk by causing dilution of urine and reducing the stay time of free stone particles in urine. The incidence of kidney stone formation is common in summer due to sweating which causes precipitation of urinary constituents. Low urinary levels of magnesium and citrate while the high levels of calcium, oxalate and uric acid can cause a higher risk of urolithiasis [3].
According to WHO, about 75% of the world's population depends upon herbal remedies due to their less adverse effects.
Herbal medicines are also known as natural medicines.

f. Antiurolithic Potential of MoS.Cr
To investigate the antiurolithic potential of MoS.Cr, calcium oxalate (CaOx) urinary crystals were induced in albino rats by using 1% ammonium chloride (AC) and 0.75% ethylene glycol (EG) for the first 5 days and then EG alone for the next 16 days in drinking water.

g. Animal Model of Urolithiasis
The effects of MoS.Cr against urolithiasis were studied by dividing the animals into different groups of matched body weight.

h. Urine Collection and Analysis
All the animals were kept in metabolic cages at the end of day 21 and day 35 for collection of urine. Fresh urine samples were collected in the morning. For urinary crystal count, 1ml of each urine sample was taken and centrifuged at 3000rpm for 5 minutes.
950µl of supernatant was discarded and remaining portion was transferred to a Neubauer chamber. The types and number of crystals were determined using a light microscope as described previously [6]. After 24 hours, Urine samples were collected.
Urinary pH and urinary volume per 24hrs were determined.
Urinary levels of uric acid, phosphorus, magnesium, calcium and total protein were determined by using commercially available kits.

i. Serum Analysis
The animals were anesthetized with Ketamine/Xylazine (10:1) at the end of the day 35. Blood was collected through retro-orbital method and allowed to clot for 15 minutes. Then, centrifuged at 4000rpm for 15 minutes and serum was separated. Serum analysis was done for the determination of different biochemical parameters, i.e., creatinine and blood urea nitrogen.

j. Kidney Histology
At the end of study, animals were sacrificed. One representative kidney from each group was removed, preserved in 10% formalin and then analyzed histologically.

k. Acute Toxicity Assay
Acute toxicity test was carried out to assess the safety of MoS.Cr on Swiss albino mice of either sex weighing (18-30g) as described previously [7]. Animals were randomly divided into different groups

l. Phytochemical Screening of MoS.Cr
The aqueous alcoholic extract (MoS.Cr) was found to be rich in alkaloids, saponins, flavonoids, phenolic contents, glycosides, tannins and coumarins.

m. Effects of MoS.Cr on Urine Parameters
In present study, urine parameters, i.e., crystal count, urine  Figure 1C). Calcium and magnesium (stone inhibitor) levels were decreased in lithogenic rats ( Figure 1E and 1F, respectively). While Uric acid, phosphorus and total protein levels were increased after the lithogenic treatment ( Figure 1D, 1G and 1H, respectively).
MoS.Cr, at the doses of 100, 300 and 500mg/kg p.o. significantly reversed and restored these urinary changes towards normal and the results of MoS.Cr were almost comparable to the results of cystone (500mg/kg), the standard drug.

n. Effects of MoS.Cr on Serum Parameters
Lithogenic treatment caused highly significant increase (p<0.001) in serum creatinine and BUN levels. MoS.Cr showed highly significant results and reduced the creatinine and BUN levels (Table 1).    Uric acid is formed from the metabolism of purine rich diet such as meat, beans, green peas, cheese and oats etc. Uric acid acts as nucleator and accelerate the process of crystals formation. It also precipitates the calcium oxalate crystals by decreasing its solubility [11]. Lithogenic treatment caused significant increase in urinary uric acid levels, which were decreased significantly after the treatment protein level to a significant extent [11]. Creatinine is a nitrogenous waste product which is formed by the natural breakdown of muscle tissues and then secreted from the blood by kidney. Levels of serum creatinine and urea indicate the efficiency of kidney. In urolithiasis, glomerular filtration rate (GFR) decreases due to renal damage and the level of nitrogenous waste product increases. Concentration of creatinine in serum was increased due to the consumption of ethylene glycol. MoS.Cr showed significant restoration of increased levels of creatinine towards normal in increasing doses [12].

Conclusions of the Study
The present study demonstrates the antiurolithiatic potential