STUDY ON IN VITRO DISSOLUTION OF CALCIUM OXALATE RENAL STONE BY SHILAJIT

Objective: This study aims to test the solubility efficiency of Shilajit in vitro for calcium oxalate renal stone. Material & Methods: A small stone was selected for the experiment. The weighed stone was suspended in 25 ml of aqueous extract of Shilajit for 72 hours with the interval of 24 hours. After each 24 hours, the stone was taken out, washed, dried and difference in weight was calculated. The whole procedure was carried out at room temperature. Results: It was found that the weight of the stone was reduced. Conclusion: Shilajit has the ability to dissolve the calcium oxalate renal stone.


INTRODUCTION
Kidney stones are one of the most painful disorders to afflict humans. In the United States, the urinary stone disease is a common clinical problem with a high risk of recurrence. One out of every ten Americans suffers from kidney stone disease sometime during their lifetime. It is the third leading cause of hospitalization in the USA and the seventh leading cause for visits to urologists. Clinical treatment of stone-related disorders is estimated to cost over $2 billion per year. Current medical treatment will not dissolve symptomatic kidney stones. Since most stones will not pass out of the body spontaneously, comminution with highintensity shock waves (SWL) has become nearly a standard procedure for stones in the upper urinary tract. However, compelling clinical data now suggest that SWL may cause acute renal injury, a rise in diastolic blood pressure, a decrease in renal function, and an increase in stone recurrence. This proposal seeks an alternative to SWL for kidney stone treatment.
Oral dissolution therapy of kidney stones with diuretic medicinal plants has been known for millennia by the indigenous people of America, 1 India, and Arabia.
The dissolution of renal calculi orally has limited indications. For example, it is used only as an adjuvant to an endourologic operation or ESWL in special situations. Chemolysis may have an important role in the management of renal stone in 2 the future. The choice of the kind of solution for the breakdown of the stone depends upon the composition of renal stone. Suby or Renacidin solution can dissolve calcium phosphates, though it is often tiresome and time-consuming process. These solutions, however, cannot dissolve the major urinary stone component, the calcium oxalate. Because of local toxicity, EDTA and other strong calcium chelator cannot be used. There are enzymes, which however can digest the organic matrix of renal stone. The indication to Chemolysis of stones is 3 rather restricted.
From a historic standpoint, in 1938 Hellstrom first dissolved a struvite stone using boric acid and permanganate. In 1943, Suby and Albright developed Suby's solution G, which could be instilled into the kidney via a nephrostomy tube or ureteral catheter. In their initial report, six patients had partial or complete stone dissolution. Mulvaney then modified this solution by adding D-gluconic acid (Hemiacidrin or Renacidin®) and published the first report dissolving infection stones in nine of 13 patients, either partially or completely. However, a report of four deaths during intrarenal irrigation led 4 to an FDA ban on renal Renacidin® use.
Serious attempts to discover a satisfactory agent for the dissolution of urinary tract calculi began when the chemical composition of most th stones was identified in the early 19 century. Since the first report of Chemolysis by Crowell in 1924, countless other urologists have used this method of eliminating all forms of urinary lithiasis. Albright et al. reported 40 years ago that a buffered solution of citric acid (pH 4.0) was effective in dissolving some phosphatic calculi, but this substance proved to be too irritating to the tissues to be clinically useful. Suby and Albright modified this solution by adding anhydrous magnesium oxide and sodium carbonate, 2 making it more acceptable to the tissues.
In Ayurveda, Shilajit is thought of as one of the miracle drugs. It is not a plant. It is not animal stuff either. It is a mineral pitch. It comes out the rocks of the Himalayas when they become warm in the summer season. It is believed that Shilajit has the therapeutic power of these great mountains.
It is called Shilajit in Sanskrit, Shilajita in Hindi, Hajar-ul-Musa in Arabic, Momiai in Persian, vegetable Asphalt in English, Asphaltum in Latin, and its botanical description is Bitumen mineral.
The Charaka Samhita states that "Stones of metal like gold etc., in the mountains get heated up by the sun and the exudates that come out of them in the form of smooth and clean gum is called çiläjatu". According to Sharma Shilajit is not produced from metals like gold rather stones containing gold produces Shilajit.
It is thought that plants like Euphorbia royleana Boiss and Trifolium repens grow near the Shilajit rocks and they are the source of Shilajit. Current research claims that bryophytes form Shilajit as species of mosses and liverworts were found in the vicinity of rock which exudes Shilajit. The mineral composition of the bryophytes is same 5 as that of Shilajit.
Laboratory analysis of Shilajit shows that it possesses a large quantity of benzoic acid, hippuric acid, and their salts. Besides, it also contains gums, 7 albuminoids, traces of resin and fatty acid. Shilajit contains fresh and modified leftovers of humus. It is 10-70% of the water-soluble part of Shilajit. This humus is mixed with plant and microbial metabolites present in the rock rhizosphere of its natural 8 environments.
Fulvic acid is the major organic matter in aqueous extract of Shilajit. It has been reported that most of the biological and medicinal properties are 9 due to it.
Externally fulvic acid has been used in the treatment of hematoma, desmorrhexis, myogelosis, arthrosis, polyarthritis, osteoarthritis, and osteochondrosis. Fulvic acid has equally been used orally in the treatment of gastritis, diarrhea, stomach 10 ulcers, dysentery, colitis, and diabetes mellitus.
Scientists have discovered recently that fulvic acid is the finest electrolyte. It helps in human enzyme production. It has a role in the utilization of vitamins. It is also a powerful antioxidant and free radical scavenger. It has a unique ability to react with both positively and negatively charged unpaired electrons and render free radicals harmless. Free radicals are either altered to new usable compounds 11 or eliminated from the body as waste.
The aqueous solution of fulvic acid has the capability of dissolving minerals and metals. The fulvic acid changes them into complex fulvic acid molecular complexes. These complexes have wide range of characteristics. Fulvic acid is a natural metallic mineral chelator and turns them into readily 12 absorbable bio-available forms. Fulvic acid can help in the movement of metal ions that are usually difficult to transport. Fulvic acid is an exceptional natural chelator and cation exchanger and is 13 essentially important in the nourishment of cells. Fulvic acid and humic acids from the Shilajit are the natural organic polyelectrolyte acids. They are 14 effective crystal growth inhibitors. Fulvic acid Ahmad: In vitro dissolution of calcium oxalate renal stone by shilajit regulates calcium carbonate growth rate inhibition. Factors included in the process are structure, molecular weight, functional group character, and functional group ionization. It seems probable that the factors in fulvic acids that increase their efficiency as calcite growth inhibitors may recommend compounds or arrangements of carboxylate groups that are valuable as beginning 15 inhibitors.
Humic acid and fulvic acid efficiency of inhibition of growth rate are due to multiple ionized carboxylate groups. These groups are directly attached to a five member ring. Growth rate inhibition of calcium carbonate may also be due to the arrangement of carboxylate groups in the fulvic 15 acid.

OBJECTIVE
This study aims to test the solubility efficiency of Shilajit in vitro for calcium oxalate renal stone.

MATERIAL & METHODS
The aqueous extract of Shilajit was prepared by dissolving 10 grams of purified Shilajit in 100 ml of deionized water. This solution was thoroughly mixed for 3-4 hours and centrifuged at 3.000 rpm for 10 min. The supernatant thus obtained was referred to as the aqueous extract of Shilajit (AES).
The dissolution was carried out following the method of Sinha et al. with minor 16 modifications. The work was carried out at Bahauddin Zakariya University, Multan, Pakistan and Multan Drug Testing Laboratory, near District Jail Multan, Pakistan in 2010-2011.
The renal stone of pure calcium oxalate composition was selected for the experiment. The stone was washed properly with distilled water. The stone was suspended in 20 ml of 0.1N NaCl solution for 24 hours then filtered and washed it with distilled water. Dried in an air oven at 80oC for 2 hours and cooled down. The stone was weighed (analytical balance, OHAUS Corp. USA) and suspended in 25 ml of the aqueous extract of Shilajit for 24 hrs. Stone was filtered, washed with distilled water, dried and weighed out. Stone was again suspended in 25 ml of aqueous extract of Shilajit for the next 24 hours. Stone was again filtered, washed with distilled water, dried and weighed out and suspended in 25 ml of each aqueous extract of Shilajit thrice for another 24 hrs. Finally, the stone was filtered and washed with distilled water and then dried and weighed out after due period. The whole procedure was carried out at room temperature.

RESULTS
Solubility efficiency of Shilajit for calcium oxalate renal calculi at different time intervals were evaluated in this work. The weight of the renal stone chosen for the experiment was 0.365 grams. The solubility difference after 24 hours, 48 hours, and 72 hours were 0.011, 0.025, and 0.035 respectively and percentage solubility was 3.01, 6.04, and 9.58 respectively as shown in Table 1 and Table 2.

DISCUSSION
From the results, it is quite apparent that weight reduction did occur. The dissolution of calcium oxalate in the presence of Shilajit may be described as due to the chelation of Ca2+ ion with the major active principle of the Shilajit such as fulvic acid as it is amongst the numerous active principles 17 of Shilajit. Various Therapeutic actions of Shilajit have been attributed to fulvic acid and 18 dialphabenzopyrone.
Fulvic acid has the ability to form complexes with mono-, di-, tri-and polyvalent metal ions. These complexes are stable and water-soluble. It can also shift the metal ions that are difficult to mobilize in normal circumstances. Fulvic acids are good natural chelators, cation exchangers and very [12][13] important in the nourishment of cells.
From the above discussion, it may be said that fulvic acid in the Shilajit might have formed a stable complex with calcium present in calcium oxalate renal stone and sequestrated it from the stone. This may be the probable mechanism of dissolution of renal calculus.
F u l v i c a c i d , a n a t u r a l o r g a n i c polyelectrolyte acid, is effective crystal growth 17 inhibitors. It may further be concluded that the carboxylic group in the fulvic acid acts as chelator of calcium.
Fulvic acid regulates calcium carbonate growth rate. Factors included in the process are structure, molecular weight, functional group 15 character, and functional group ionization. The same phenomenon of sequestrating the calcium from the renal calculus may be taking place in the dissolution process. Therefore it is gathered that the carboxylic group of the fulvic acid complexes with the calcium of the renal calculus and it is dissolved. This finding is corroborated by the work done by Curtis et al. who established that Anticolana Valley (Fiuggi) water contains humic and fulvic acid. Keeping in view this fact, the ability of this 19 water to dissolve kidney stone in vitro was studied. The Anticolana Valley (Fiuggi) water, distilled water, and tap water were used in the study. A specially designed Perspex apparatus was used in the experiment. Each stone was subjected to a water flow of 2 liters /24 h. It was found that the ability of Anticolana Valley water to dissolve the human as well synthetic stone was very high as compared to that of distilled water. And distilled water was found to be more effective than that of tap water.
Fraioli et al. continued study on Fiuggi water and after an in vivo study on human beings, he found that after drinking the said water calcium oxalate monohydrate was either strongly reduced or sometimes even removed from the urinary 20 sediment. In this way, the risk of oxalate calculosis was greatly reduced. Organic molecules belonging to fulvic acid family are present in the fiuggi water.
These acids can make complexes with calcium ions. These acids form a film on the calcium oxalate and interact preferentially with the crystal lattice of the calcium oxalate monohydrate. These behave as pumping systems by linking the calcium ion, demolishing the crystal lattice and dissolving calcium and oxalate ions.
Calace et al. isolated humic acid from the anticolana valley (fiuggi) water purified and 21 characterized it. Only fulvic acids were found. These are mainly composed of aliphatic chains, made of six -CH2O-groups and contain the number of carboxylic acids group responsible for their metal complexing capacity.

CONCLUSION
Shilajit has the ability to dissolve calcium oxalate renal stone. Study on larger level is however required to establish the results.