Effects Of An Aqueous Extract FromPhyllantus Niruri On Calcium Oxalate Crystallization In Vitro

Original Paper

Effects of an aqueous extract fromPhyllantus niruri on calcium oxalate crystallization in vitro

M. E. Barros1, N. Schor1 and M. A. Boim1, 2

(1)

Nephrology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil

(2)

UNIFESP Renal Division, Rua Botucatu 740, 04023-900 São Paulo, SP, Brazil

M. A. Boim

Email: mirian@nefro.epm.br

Received: 10 April 2002Accepted: 10 October 2002Published online: 21 January 2003

Abstract

Phyllanthus niruri is a plant used in Brazilian folk medicine for the treatment of urolithiasis. It was previously observed that P. niruri shows no toxicity, potentially increases calculus voiding by stone forming patients and inhibits the endocytosis of calcium oxalate (CaOx) crystals by MDCK cells. In addition, in a rat model of urolithiasis it reduced calculus growth. In the present study, we evaluated the effect of an aqueous extract ofP. niruri on CaOx crystallization in vitro. CaOx precipitation was induced by the addition of 0.1 M sodium oxalate to unfiltered urine samples from Wistar rats (n=14) and normal humans (n=18) in the presence or absence of P. niruri extract (0.25 mg/ml of urine). The presence of CaOx crystals was evaluated immediately and 24 h later. In vitro crystallization of human urine produced typical mono- and dihydrated CaOx crystals, but only a few typical CaOx crystals were found in rat urine. The presence of P. niruri extract did not inhibit CaOx precipitation and even more crystals were obtained, although they were significantly smaller than those in the control urine. Crystal aggregation observed 24 h after crystallization was also inhibited by P. niruriextract. The results showed an inhibitory effect of P. niruri extract on CaOx crystal growth and aggregation in human urine, suggesting that it may interfere with the early stages of stone formation and may represent an alternative form of treatment and/or prevention of urolithiasis

Keywords

Calcium oxalate In vitro crystallization Phyllanthus niruri Renal stone Urolithiasis Natural products

Introduction

Urinary stones affect 10–12% of the population in industrialized countries [27]. Their incidence has been increasing over the last years while the age of onset is decreasing [10]. In addition, the recurrence rate is high, being more then 50% after 10 years [28, 29]. In spite of substantial progress in the pathophysiology and treatment of urolithiasis, there is no satisfactory drug to use in clinical therapy. Thus a drug for the prevention of this disease or its recurrence would be of great interest.

Phyllanthus niruri is a plant belonging to the Euphorbiaceae family, which have a worldwide distribution. It is used in Brazilian folk medicine by patients with urolithiasis [18, 21]. Many components of P. niruri have been identified, including groups of active substances such as alkaloids, tannin, lignans, phenols, steroids, flavanoids, triterpenes, as well as ricinoleic acid, niruside, and phylitate [4]. However the components involved in lithiasis prevention are not known. We have been evaluating the potential effect of P. niruri in the treatment of urolithiasis over the last few years. Experimental and clinical studies have demonstrated that P. niruri has no acute or chronic toxicity and preliminary data suggest effects which promote stone elimination in stone forming patients [25]. Moreover, oral administration of P. niruri aqueous extract to rats induced an inhibitory effect on vesical calcium oxalate (CaOx) crystal growth, which was associated with a reduction in the urinary excretion of glycosaminoglycans and with an increase in the content of these macromolecules in the calculi compared with untreated animals [11]. Also, P. niruri significantly reduced the endocytosis of CaOx crystals in MDCK cells in culture [5]. Despite the beneficial effect of P. niruri observed in vivo in rats and humans, its mechanism of action is not fully understood.

Most kidney stones contain calcium oxalate [9], and the formation of urinary calculi involves a CaOx crystallization process that includes nucleation, growth and the aggregation of crystals [15, 16]. Thus, to better understand the role of P. niruri in urinary stone formation in the present study, we evaluated the effect of an aqueous extract of this plant on CaOx crystallization induced in vitro.

Materials and methods

An aqueous extract of P. niruri was obtained from the whole plant, as occurs in popular medicine. The plant was grown at the Experimental Center of the Universidade Estadual de Campinas, São Paulo. Plant samples were dried at 50°C for 2 months in a ventilated room. After drying, samples were ground in a mechanical mill and used for tea preparation (5% w/v). The infusion was stirred for 30 min at 72°C and then vacuum filtered, concentrated and lyophilized.

CaOx crystallization was induced in the urine obtained from normal humans and rats. Isolated human urine samples were obtained from six healthy subjects, three males and three females, with no personal or family history of kidney stone disease. Urine was collected on three different occasions from each individual, with an interval of at least 15 days between each sampling time (n=18). Urine samples collected over 24 h were obtained from normal, adult Wistar rats (n=14) in a metabolic cage. Rats were maintained on standard chow and tap water during the collection period. Human and rat urine samples were centrifuged at 5,000 rpm (4,815 g) for 8 min, the supernatant was then transferred to a clean tube, and the pH was adjusted to 6.0.

Experimental protocol

CaOx precipitation was induced by adding 40 μl of 0.1 M sodium oxalate per ml of urine (corresponding to 0.536 mg), every 30 min (0, 30, 60 and 90 min) under shaking at 37°C, resulting in a final concentration of 2.14 mg/ml of urine. Each urine sample was divided into two aliquots, one of which was used as a control (crystallization without P. niruri extract) while in the other CaOx precipitation was induced in the presence ofP. niruri extract, which was added to the sample 30 min before the crystallization process. Lyophilized P. niruri extract was resuspended in distilled water (25 mg/ml), filtered through a 0.22 μm filter, and used at a final concentration of 0.25 mg/ml urine, based on a dose-response curve.

The present protocol was approved by the Ethics Committee of the Universidade Federal de São Paulo.

Analysis of crystals

The crystals obtained were analyzed immediately and 24 h after the crystallization process. The semiquantitative analysis of crystals was estimated by turbidity [26]. After crystallization, 100 μl aliquots were loaded onto a 96 well microplate and the absorbance was measured with a plate reader

...