Phytopharmacological Evaluation of Antiurolithiatic Activity of Grewia Flavescens and Macrotyloma Uniflorum

Abstract

Urolithiasis (commonly known as kidney stone, renal stone), the formation of stone in the human urinary system is a common and painful disease. In all over the world, ap proximately 4-15 % of the human population suffer from urinary stone. Since urolithiasis is a multifactorial disease, its etiology is very complex and highly unpredictable. Manage ment of urolithiasis mostly depends on stone size and its location in the urinary system. In most of the cases stones are removed by surgical treatment, but unfortunately stone recurrence was observed about 50 % after removal by surgical treatment or may cause serious metabolic and disease complications to patients and they also impose a load of costs to the healthcare system. So there is an increased interest in the alternate sup porting therapy for the management of urolithiasis using natural products and ayurvedic medicines. In Ayurveda, many traditional plants are reported to be used in the treatment of urolithiasis. In India, Grewia flavescens (family: Tiliaceae) roots and Macrotyloma uni- florum (family: Fabaceae) seeds are commonly used as therapeutic agents in renal stone problems. However, till date there is no scientific evaluation report for these plants to be effective in treating renal stone. So, intends of present study to establish the scientific rationality of aqueous extract, methanolic extract and its ethyl acetate, n-butanol and aqueous fraction of G. flavescens roots and M. uniflorum seeds as a anti-urolithiatic ac tivity using in vitro crystallization assay of calcium oxalate (CaOx) crystals, viability test on MDCK cell line and in vivo sodium oxalate (N aOx) (Preventive model) and ethylene glycol (EG) (Curative model) induced urolithiasis in the albino Wistar rats. The study was further aimed to identify the phytoconstituents of extracts which possessing biological activity using HPTLC and HPLC methods. All extracts and fractions at different con centrations (250-2000 µg/ml) were subjected for in vitro activity like nucleation, growth of CaOx crystals, crystal aggregation and CaOx crystals dissolution. The nucleation and crystal growth assay were performed using different concentrations of N aOx and CaCl2 (2-10 mmol/ml) for nucleation and (2-3.5 mmol/ml) for crystal growth and evaluate the inhibitory effect of extracts and fractions at different concentration level of oxalate and calcium. CaOx crystal aggregation and crystal dissolution assays were performed and compared with the effects observed in standard cystone. Four doses of extracts and frac tions (100, 200, 400 and 800 mg/kg) were studied for the diuretic activity and NaOx (70 mg/kg, i.p for 7 days) induced urolithiasis and two higher doses (400 and 800 mg/kg) in EG (0.75 % v/v in water for 28 days) induced urolithiasis (Preventive study) in Wistar albino rats. Determination of volume of urine and urinary pH, animal body and organ weight, estimation of various parameters like oxalate, calcium, urea, creatinine, uric acid, phosphate, magnesium and citrate levels in urine, serum and kidney homogenate and esti mate antioxidant parameter in kidney, histology study of kidney for CaOx deposition and cell damage in cell tissue, urinary crystal size were done at the end of experiment. The results were compared with a Cystone (standard herbal product,available in the market) treated group animal. All extracts and fractions were significantly more effective than cystone at inhibiting the nucleation rate and crystal growth when evaluated at different concentration levels of oxalate and calcium and inhibition of CaOx crystal aggregation and crystal dissolution were observed in dose dependent manner in the in vitro experi ments. MDCK cells line study indicated that extracts and fractions were prevented the oxalate induced toxicity and reduced the LDH release on cell line. In vivo study showed that extracts and fractions have significant diuretic activity, which was similar to stan dard drug hydrocholrthiazide. Co-administration of extracts and fractions with NaOx for 7 days has showed significantly (p < 0.001) increased in the urine volume and urinary pH, calculus inhibitors levels like citrate and magnesium, increased creatinine clearance and decreased the calculus promoters levels like oxalate, calcium, phosphate, urea, and uric acid. Similarly in preventive study all extracts and fractions showed prevention of crystal growth and decreased the stone promoters levels and increased glomerular activity of the kidney. Histopathology of the kidney showed significant improvement after treat ment with extracts/fractions of G. flavescens roots and M. uniflorum seeds in N aOx and EG models. Evaluation of biological parameters indicated that ethyl acetate fraction was more effective followed by aqueous, methanolic extracts and n-butanol fraction. HPTLC and HPLC data indicate that extracts contain the gallic acid, rutin, quercetin, catechin, diosgenin as a major phytoconstituents which may be responsible for the antilithiatic activity. These results indicate that extracts and fractions showed significant activity in kidney stone (urolithiasis) which might be due to its diuretic activity, inhibitory effects on CaOx crystal formation, dissolution of CaOx crystals and its ability to increase the levels of crystals inhibitors and decrease the levels of crystals promoters of urolithiasis. How ever, further studies are needed to isolate and characterize antiurolithiatic compounds in pure forms. The study also deserves toxicological evaluation and clinical trials to develop novel herbal drugs for urolithiasis.