Evaluation of the Anti-Kidney stone (Anti-urolithiasis) Activity of Natural Products, and Study of their Mechanism of Action

Abstract

The key objective of the present project was to study the preventive and curative effects of some natural products in a rat model of kidney stone disease. The first part of this thesis includes results of in vitro studies in which Boerhavia diffusa (Linn.) and Bryophyllum pinnatum (Lam.) exhibited anti-kidney stone activity by inhibiting the crystallization of calcium oxalate monohydrate crystals. A well known natural product, caffeic acid (1), was also evaluated for its in vitro antiurolithic activity, which showed a protective effect against the formation of calcium oxalate monohydrate crystals. Compound I has been obtained from various medicinal plants, including B. pinnatum (Lam.). In the second part, in vivo studies showed that B. pinnatum (Lam.) is a potent antiurolithic herb in ethylene glycol-induced urolithic rats. Twenty four hours urine analysis of the preventive group showed significant effects of herbal treatment on the urinary citrate (**p < 0.01), oxalate (**p < 0.01), calcium (*p < 0.05), magnesium (***p < 0.001), urea (***p < 0.001), and creatinine (**p < 0.01) levels. The curative treatment of rats with B. pinnatum extract also significantly regulated the altered urinary levels of citrate (***p < 0.001), oxalate (**p < 0.01), calcium (***p < 0.001), magnesium (***p < 0.001), urea (***p < 0.001), and creatinine (**p < 0.01). Similarly, caffeic acid (1) significantly regulated the pathologically altered levels of citrate (*p < 0.05) and oxalate (**p < 0.01) in the urine. Histopathological studies also revealed the protective effects of Bryophyllum pinnatum (Lam.) extract, and caffeic acid (1) on renal architecture. The third part comprised molecular studies in which renal tissues of rats from different groups were processed for RNA isolation, cDNA synthesis, reverse transcriptase-polymerase chain reaction (RT-PCR), agarose gel electrophoresis, and real-time polymerase chain reaction (RT-qPCR). B. pinnatum (Lam.) extract increased the expression of prothrombin fragment 1 (*p < 0.05), bikunin (*p < 0.05), and Tamm-Horsfall genes (*p < 0.05), while it decreased the renal expression of osteopontin gene ((*p < 0.05), in comparison to the pathological control. In case of caffeic acid (1), prominent effects were observed where the expression of bikunin (*p < 0.05), prothrombin fragment 1 (*p < 0.05), and Tamm-Horsfall (**p < 0.01) increased significantly, while that of osteopontin gene (**p < 0.01) significantly decreased as compared to the pathological control. The results of western blotting also indicated that caffeic acid (1) has the potential to affect the expression of prothrombin fragment 1 at the protein level. The outcomes of this study could help to understand the possible molecular mechanism behind the antiurolithic effects of related natural products, and to investigate their therapeutic potential. A bioassay-guided isolation of secondary metabolites from these plants needs to be conducted in order to identify compounds responsible of the antiurolithic activity.