Phytochemical Characterization and Evaluation of Anti-Urolithiatic Activity of Marketed Herbal Formulation

Abstract

Urolithiasis (also referred to as kidney stones or stones in the kidneys) occurs when stones form in the human urinary system. There are about 4-15% of people worldwide who suffer from urinary stones. As, Urolithiasis is a multifactorial disease, and therefore its etiology is very highly complex and also unpredictable. Treatment of this depends primarily on the size of the stone and its location in the urinary system. A majority of the stones are removed surgically, but recurrences are possible approximately 50 years after removal, leading to serious metabolic and disease complications. The medical costs associated with the system are high. Consequently, natural products and Ayurvedic medicines are becoming more attractive for use in the management of urolithiasis. The Ayurvedic system of medicine reports that many traditional plants are used to treat urinary calculi. As a treatment for renal stones, Pashanbhed - Bergenia ligulate, Boerhavia diffusa, Crataeva nurvala, and Tribulus terrestris (Gokshur) are commonly used in India. In both Formulation 1 and Formulation 2, these are the common plants used. A significant number of phenolics, flavonoids, and triterpenoids are present in these plants, and they are used as a treatment for urolithiasis. A simple and precise HPTLC method was utilized to identify the various bioactives contained in both Formulation 1 and 2. A TLC analysis of silica gel 60F254 plates was used for the determination of quercetin, lupeol, rutin, and gallic acid. An ultraviolet densiometric scan was performed using the CAMAG TLC scanner 3 at wavelengths of 254 nm and 366 nm on the plates. Based on preliminary screening using a mobile phase containing a variety of polar and non-polar components, toluene, ethyl acetate, methanol, and formic acid in the ratio of (6.6: 6.6:5.3:1.3) were selected for use as a mobile phase for Quercetin and Gallic acid in formulation 2, Tol:EA:FA(5:4:1) for Gallic acid in Formulation 1, Butanol:AA:Water (7:7:7) for rutin in both formulations, and Tol:EA:FA:MeOH(7:6:1:2) for lupeol in Formulation 2. Rf values for formulation 1 and formulation 2 were 0.84 for quercetin, 0.85 and 0.78 for rutin, 0.69 and 0.75 for gallic acid, 0.81 for lupeol, respectively. Based on HPTLC results, Formulation 1 and 2 contains gallic acid, rutin, quercetin, and lupeol as major phytoconstituents that may contribute to the antilithiatic activity.Both formulations were tested in vitro at different concentrations (200-1000μg/ml) for nucleation and crystal aggregation. In order to evaluate the inhibitory effect of sample at different concentration levels of oxalate and calcium, different concentration levels of CaCl2 (5 mmol/l) and Na2C2O4 (7.5 mmol/l) solutions were used for nucleation and crystal aggregation. A CaOx crystal aggregation assay was performed and the results were compared to those of normal cystone. Using different concentrations of samples after 60 min of incubation, it was found that cystone inhibited nucleation activity the most with 77.9 % at 1000 μg/ml, whereas the sample for Formulation 1 showed 68.8 % and Formulation 2 showed 72.8 %, which are almost similar results to cystone's. Based on the inhibitory aggregation effect at different concentrations after 60 min of incubation, Cystone had 32.958 % inhibition, Formulation 1 had 33.803 % inhibition at 60 min, which is similar to Cystone and Formulation 2 had 26.76 % inhibition. Crystallization of calcium oxalate was inhibited by the increased concentration of extracts