Pharmacological basis for the medicinal use of berberis vulgaris and bergenia ligulata in urolithiasis

Urolithiasis is world-wide, sparing no geographical, cultural or racial groups. Currently available stone removal techniques are relatively safe and effective in majority of the cases but for a large proportion of the world population with urinary stones, it is no longer an option of choice mainly on account of lack of affordability. Recurrent nature of urolithiasis makes it a chronic condition requiring continuous management. The prophylactic agents used clinically are primarily aimed to correct the underlying metabolic disorders but the evidence for their effectiveness is less convincing. Accumulating scientific evidence of their safety and efficacy has made traditional medicines, mainly the herbs, a well-accepted alternative therapy which is at the same time the treatment of choice for up to 70Vo of the world population even today. This study was undertaken to evaluate the selected medicinal plants for their antiurolithic effect on an animal model of calcium oxalate (CaOx) urolithiasis and to elucidate possible mechanism(s) of action in order to rationalize their medicinal use in urinary stone disease. The crude aqueous-metabolic extracts of Berberis vulgaris root bark and Bergnia ligulata root, their petroleum spirit, n-butanol and aqueous fractions and the pure compounds, bergenin and berberine, respectively were studied using in vitro and in vivo methods. In the metastable solutions of CaOx, Berberis vulgaris root bark extract (Bv.Cr) decreased the rate of CaOx crystal nucleation and aggregation as well as crystal count and exhibited antioxidant effect against lipid peroxidation induced in rat kidney homogenate by ferrous-ascorbate system and 1,1-diphenyl-2- picrylhydrazyl (DPPH) free radical. When tested on rabbit urinary bladder strips, Bv.Cr caused spasmolytic effect against high K+ (80 mM) and carbachol (1 µM)- induced contractions mediated through dual blockade of muscarinic receptors and calcium channels. In an animal model of urolithiasis, developed in male Wistar rats by adding O.757o ethylene glycol in drinking water, Bv.Cr (25-50 mg/kg) inhibited CaOx crystal deposition in renal tubules as well as eliminated pre-deposited crystals at 50 mg/kg. It also protected against the deleterious effects of lithogenic treatment including polydipsia, polyuria, weight loss, impaired renal function and development of oxidative stress. Activity-guided fractionation revealed the distribution of antioxidant activities among both n-butanol and aqueous fractions, CCB effect mainly in the aqueous fraction and anticholinergic activity in butanol fraction, whereas, the in vitro crystal inhibition and in vivo antiurolithic activities were concentrated in the aqueous fraction. The main plant constituent, berberine exhibited in vitro antioxidant effect against lipid peroxidation and DPPH free radical and found more potent than Bv.Cr in these assays. Berberine relaxed both carbachol (CCh) and high K+-induced spasms in rabbit urinary bladder mediated through a combination of anticholinergic and week CCB activities. In the animal model of CaOx urolithiasis, berberine (10 mg/kg) prevented as well as cured the CaOx uroliths and protected against associated changes. In naïve rats, Bv.Cr (50 mg/kg) and berberine (10 mg/kg) did not cause any change in urine volume, pH and composition. Bv.Cr and berberine, at 50 and 10 mg/kg, respectively, increased total antioxidant status of serum, without causing any renal or hepatic toxicity or affecting the haemoglobin level and erythrocyte sedimentation rate. In the in vitro studies, the Bergenia ligulata crude extract (Bl.Cr) decreased the rate of CaOx crystal aggregation as well as crystal formation induced in the metastable solutions of CaOx. It also exhibited antioxidant effect against in vitro lipid peroxidation and DPPH free radical. When tested on rabbit urinary bladder strips, Bl.Cr caused spasmolytic effect against CCh and high K+-induced contractions mediated through dual blockade of calcium channels and muscarinic receptors. Bl.Cr caused diuretic effect in rats mediated by a saluretic effect. In the animal model of urolithiasis, Bl.Cr dose-dependently (5-10 mg/kg) prevented CaOx crystal deposition as well as eliminated the crystals pre-deposited in the renal tubules at the dose of 10 mg/kg. It also protected against the other effects of lithogenic treatment including polydipsia, polyuria, weight loss, impaired renal function and development of oxidative stress in the kidneys. In naive rats, Bl.Cr (10 mg/kg) increased urine output, Mg++ excretion, improved serum total antioxidant status and found devoid of any adverse effect on liver and kidneys functions and haematological parameters. Activity-guided fractionation revealed the distribution of antioxidant activities among both n-butanol and aqueous fractions, concentration of CCB effect mainly in the aqueous fraction and anticholinergic activity in butanol fraction, whereas, the in vitro crystal inhibition, diuretic effect and in vivo antiurolithic activities were concentrated in the aqueous fraction. Bergenin, the commercially available pure compound of Bergenia ligulata, was found devoid of significant activity in any of these studies. These data suggest the presence of antiurolithic effect in Berberis vulgaris, berberine and Bergenia ligulata against CaOx urolithiasis. The effect of Berberis vulgais and berberine was mediated possibly through a combination of CaOx crystal inhibition and antioxidant activities and Bergenia ligulata through CaOx crystal inhibition, antioxidant and diuretic effects and increase in urinary Mg* content. Both $e plant extract and berberine exhibited spasmolytic effect mediated through combination of calcium channels and muscarinic receptor blockade, which may also add to their overall antiurolithic potential. Activity-guided fractionation revealed the separation of antiurolithic activities of Bv.Cr and Bl.Cr in their respective aqueous fractions. This study rationalizes the medicinal use of Berberis vulgaris root bark and Bergenia ligulata root in urolithiasis and these data may help designing future studies on Berberis vulgaris, Bergenia ligulata and berberine to establish their efficacy and safety for clinical use