Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats

Journal of Ethnopharmacology 129 (2010) 38–45

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Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats S. Bouanani a,b , C. Henchiri c , E. Migianu-Griffoni b , N. Aouf a , M. Lecouvey b,∗ a b c

Département de Chimie, Laboratoire de Chimie Bioorganique, Université Badji Mokhtar, Annaba, Algeria Equipe Chimie Bioorganique et Bionanomatériaux (C2B), Laboratoire CSPBAT, FRE 3043 CNRS, Université Paris 13, Bobigny, France Département de Biochimie, Laboratoire de Biochimie Appliquée et Microbiologie, Université Badji Mokhtar, Annaba, Algeria

a r t i c l e

i n f o

Article history: Received 29 July 2009 Received in revised form 27 January 2010 Accepted 27 January 2010 Available online 4 February 2010 Keywords: Algerian herbal medicine Paronychia argentea Calcium oxalate Nephrolithiasis Aqueous and butanolic extracts

a b s t r a c t Aim of the study: Renal protection and antiurolithiasic effects of two extracts of Paronychia argentea (PA), a traditional Algerian plant commonly known as Algerian tea, were evaluated. This study was carried out to determine whether the aqueous extract (APA) or the butanolic extract (BPA) of aerial parts could prevent or reduce calculi aggregation in experimental calcium oxalate (Ox) nephrolithiasis in Wistar rats. Materials and methods: The two extracts (APA and BPA) were administrated orally and daily, during 28 days to nephrolithiasic treated rats at the dose of 250, 500 mg/kg b.w. and 10, 20 mg/kg b.w. respectively. Body weight, renal index, liver index, serum level of creatinine, uric acid, urea, K+ , Ca2+ , Mg2+ , Na+ and transaminase (alanine aminotransferase, ALT; aspartate aminotransferase, AST), phosphatase alkaline activity (PAL) were evaluated following the 28 days treatment in rats. In addition histopathological changes in kidney and liver were stained in hematoxylin eosin (HE). Results: The effect of the extracts could be advantageous in preventing urinary stone retention by reducing renal necrosis and thus inhibit crystal retention. In contradiction with APA, the two doses of BPA attenuated elevation in the serum creatinine (p < 0.01) and blood urea levels (p < 0.01) (nephroprotective effect). However, the increase in ALT (27%) and PAL (31–51%) serum levels and in the relative liver weights (p < 0.01) in the groups treated with doses of APA may indicate that this extract has not a hepatoprotective effect against oxalate toxicity. Conclusions: The presented data indicate that administration of the butanolic extract of aerial parts to rats with NaOx induced lithiasis, and reduced and prevented the growth of urinary stones in experimental calcium oxalate nephrolithiasis in Wistar rats. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Urinary stone is a common disorder with a recurrence rate of 70–81% in male and 47–60% in female (Smith and Guay, 1992). Calcium oxalate (CaOx) represents up to 80% of analyzed stone (Perien and Perien, 1968). Kidney stone formation is a complex process that results from a succession of several physicochemical events including supersaturation, nucleation, growth aggregation and retention within the renal tubules (Khan et al., 1982; Laroubi et al., 2007). Among the used treatments, there are extracorporeal shock wave lithotripsy (ESWL) and drug treatment which revolutionized urological practice and almost become the standard procedure for eliminating kidney stones. However, in addition to traumatic effects of shock waves, residual stone fragments persist and infec-

∗ Corresponding author at: Université Paris 13, 74 Rue Marcel Cachin F-93017 Bobigny, France. Tel.: +33 148 38 77 09; fax: +33 148 38 76 25. E-mail address: [email protected] (M. Lecouvey). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.01.056

tion could occur. Moreover, ESWL may cause acute renal injury, a decrease in renal function (Kishimoto et al., 1986), haemorrhage and hypertension (Selvam et al., 2001). Therefore, it is worthwhile to look for an alternative to these means by using medicinal plants or phytotherapy. A number of vegetable drugs have been used in many parts of the world for the treatment of urolithiasis (Grases et al., 1995; Araújo Viel et al., 1999; McHarg et al., 2003; Atmani et al., 2004; Laroubi et al., 2007). Paronychia argentea (PA) is a plant from the family of illecebraceae, popularly known as Arabic tea. Medicinal uses of the aerial parts are indicated in Algerian popular medicine as diuretic and for the treatment of the renal diseases, especially as antiurolithiasis (Beloued, 1998; Zama et al., 2007), hypoglycaemic activity (Afifi et al., 2005; Carmona et al., 2005) and antimicrobial activity (Al-Bakri and Affifi, 2007). In Portugal, PA is used as analgesic, in stomach ulcer, anorexia and flatulence (Ferreira et al., 2006). Despite the interesting results obtained from the pharmacological studies and their potential therapeutic usefulness, no pharmacological study or toxicological investigation of this species has been reported.

S. Bouanani et al. / Journal of Ethnopharmacology 129 (2010) 38–45

In this work, we evaluated the effect of the oral treatment with aqueous (APA) and butanolic (BPA) extracts on the growth and retention of the experimental calcium oxalate lithiasis stones induced by intraperitoneal injection of sodium oxalate in rats. 2. Materials and methods 2.1. Reagents n-Butanol, petroleum ether, dichloromethane were purchased from Carlo Erba. Sodium oxalate was purchased from Sigma–Aldrich. 2.2. Plant materials Aerial parts of Paronychia argentea were collected by S. Bouanani in the region of Taref, located at 60 km from the city of Annaba (Algeria) in April 2007. It was confirmed by Pr. G. Debelair and identified by Dr. G. Saladin who is a botanist at the Limoges University (France). Voucher specimen was deposited in the Life Science Department of the Limoges University under reference Pa1.Ill.2007. 2.3. Preparation of plant extracts The aerial parts were dried at 40 ◦ C, powdered in a mill and two extracts were prepared. A butanolic extract was prepared by a maceration of 300 g of this material in methanol/water (80/20) for 72 h. The extract was evaporated under vacuum and then dissolved in hot water. Chlorophyll and lipophilic parts were removed by extraction respectively with petroleum ether and dichloromethane. The water suspension was then extracted with n-butanol and evaporated giving a butanolic extract BPA (3.9 g). The aqueous extract was prepared by infusion of PA (50 g) in boiled water for 10 min, filtered and lyophilized (APA, 2.5 g). The two extracts were subjected to toxicological and antiurolithiasic tests. 2.4. Oral acute toxicity In this assay, Wistar female and male rats were divided into nine groups of 10 rats weighting 120–180 g. These rats were obtained from the animal facility of the Pasteur Institute of Algeria and kept at room temperature. Animals were fed by a standard French diet (U.A.R.210) and allowed free access to water. Toxicological tests were evaluated by the preparation of different concentrations of the two extracts which were administered orally (250, 500, 1000, 1500, 2000 mg of APA/kg and 5, 10, 20, 40 mg BPA/kg dissolved in water). Water was administrated to the ninth group which was used as control. All the animals were observed for that appearance of signs of toxicity or death during 14 days.

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divided into eight groups of six animals, acclimatized in cages for 2 weeks before the test. 2.5.2. Urolithiasis induction Oxalocalcic lithiasis were induced by an intraperitoneal injection of sodium oxalate (NaOx) (7 mg/100 g of body weight) (Khan et al., 1982). Six hours after injection, animals with urinary crystal (+ and ++) were selected for the experiment. The animals were divided in eight groups of six rats each: Group I: normal rats control. Group II: lithiasic not treated rats. Group III: normal rats treated with APA (500 mg/kg b.w. (body weight)). Group IV: normal rats treated with BPA (20 mg/kg b.w.). Group V: lithiasic rats treated with APA (250 mg/kg b.w.). Group VI: lithiasic rats treated with APA (500 mg/kg b.w.). Group VII: lithiasic rats treated with BPA (10 mg/kg b.w.). Group VIII: lithiasic rats treated with BPA (20 mg/kg b.w.). Each animal was weighted every day during all the experimental period (28 days) and observed daily for clinical signs and mortality. 2.6. Body weight of rats The body weight of each rat was estimated during the experimental period, once before the treatment and every day during the treatment for the respecting of the dose. The relative body weight (RBW) of each animal was then calculated as follows: RBW =

ABT (g) × 100 IB (g)

where ABT is the absolute body weight at one time interval and IB is the body weight of rat on the beginning of the treatment. 2.7. Biochemical and haematological analysis On the 28th day of the experimental period, all the animals were sacrificed under anaesthesia. Blood samples were collected from the jugular vein of each animal and submitted to biochemical and haematological tests. Blood samples were collected into two tubes (heparinized and dry no heparinized centrifuge tubes). For the hepatic function, serum alkaline phosphatase (PAL), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined enzymatically by standard methods using kits with an automate HITACHI 902. While for the renal function, serum urea, uric acid and serum creatinine levels were estimated by commercially available Roche kits. The serum value of Mg2+ , Ca2+ , K+ and Na+ were evaluated (9180 Electrolytes analyzer). The heparinized blood was used for a haematological analysis which was determined by blood autoanalyzer (XT 2000 automate). 2.8. Vital organ relative weight of some rats

2.5. Sub acute oral toxicity in rats 2.5.1. Animals and treatment The care and handling of the animals were in accordance with the internationally accepted standard guidelines for use animal. The protocol was approved by the institutional committee on animal care of the Pasteur Institute following the Guide for the Care and Use of Laboratory Animals (ILAR, Institute of Laboratory Animal Resources, National Academy Press, Washington, DC, 1996). Forty-eight male Wistar Albino rats weighting 120–180 g were obtained from the animal facility of the Pasteur Institute of Algeria and kept at room temperature. Animal were fed by a standard French diet (U.A.R.210) and allowed free access to water. They were

The animals were fasted 12 h before being sacrificed under ether anaesthesia. After taking the blood, the abdominal cavity of each animal was opened and organs (liver, spleen and kidneys) were quickly removed, cleaned with ice-cold saline solution and weighted. The relative organ weight (ROW) of each animal was then calculated as follows: ROW =

ABW × 100 BWS

where ABW is the absolute organ weight and BWS is the body weight of rat on day of sacrifice.

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2.9. Histopathological analysis Fragments of the liver and kidneys were subsequently fixed in a 10% solution of buffered formalin (pH 7.4) and enclosed in paraffin. 4 ␮m sections were obtained and colored with the hematoxylin eosin for evaluation under optical microscope. We note −, +, ++ and +++ for normal feature, little, appreciable and severe necrosis respectively. 2.10. Statistic evaluation The values were expressed as mean ± S.E.M. (standard error of the mean). The statistical analysis of data was realized by analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparison test using 5% level of significance. The statistical package used was MINITAB 13.4. 3. Results 3.1. Acute toxicity No toxic symptoms or death was observed in any animals during the 14 consecutive days of the treatment. Therefore, the acute minimum lethal dose (LD50 ) of the aqueous and the butanolic extracts of aerial part for Wistar rats are higher than 2000 mg/kg b.w. and 40 mg/kg b.w. respectively. 3.2. Sub acute toxicity 3.2.1. Effect on the body weight Body weight increased in all the groups (animals treated and control) (Table 1). But it has been observed a gain of body weight higher in the normal control group than in treated groups. This effect was more pronounced in the group treated with APA 250 mg/kg b.w. Variation of the body weight during the experimental time was 65.55% for the normal control group I whereas for those treated with APA 250 (V), APA 500 (VI), BPA 10 (VII), BPA 20 (VIII), control group APA 500 (III), control group BPA 20 (IV) and lithiasic nontreated group (II), the values were 56.42%, 30.95%, 33.52%, 37.92%, 35.01%, 43.88% and 10.39% respectively. Table 1 reveals that all the groups treated with sodium oxalate lost weight during the days after the beginning of treatment. The untreated group lost more weight than the treated groups. This difference was significant (p < 0.001). During the period of treatment, the body weight of rats treated and control were increased in the first week. We noted a decrease of the body weight in group II (the untreated group) which persisted to the second week. It was only in the 3rd and the 4th week that a gain in the body weight of

these animals was observed (7.50% and 10.39% respectively). But this elevation was still not significant when it was compared with the control group. In day 28, all the exhibited animals gained in body weight, whereas only the animals treated with 250 mg/kg b.w. (V) and control animals treated with BPA 20 mg/kg (IV) showed a significant increase in the RBW in the 2nd week until the 4th week when compared to lithiasic group (II). 3.2.2. Effect of the two extracts on the biochemical and haematological parameters in rats Data from the clinical chemistry and haematology are respectively shown in Tables 2 and 3. The data indicate that the serum urea and creatinine were remarkably increased in untreated group II (Table 2, p < 0.01 and p < 0.05 respectively). The administration of the butanolic extract restored value of urea to normal value when compared with the control. However, the aqueous extract restored urea only at the higher dose (500 mg/kg) (group V). But for creatinine, there is no significant difference in all groups treated when compared with the normal groups. This elevation of urea in the group treated with 250 mg/kg of APA is not associated with the elevation in creatinine. We noted a rise in protein levels only in the animals of the untreated and the APA 500 mg/kg groups. The two extracts decreased kaliemia in normal treated groups (III and IV) when compared to the normal control group (p < 0.01), but only the aqueous extract restored kaliemia to normal. No significant difference was observed in the values of uric acid, calcium, sodium or magnesium. With regards to plasma enzyme determination, an increase in the values of PAL activity was detected in the untreated group. This activity was restored only in the group treated with the higher dose of BPA. For ALT the elevation was observed only in the untreated group and the group treated with APA 500 mg/kg b.w. (p < 0.05). However, we noted differences in the values of AST but there were not statistically significant. The haematological tests revealed a significant increase in the red blood cells in the groups treated by APA (p < 0.01) associated with elevation in the packed cell volume and haemoglobin level. We noted no significant difference in the platelet level and in white blood cell count. The results showed a decrease in the lymphocyte percentage in the untreated group but it was not statistically significant (Table 3). 3.2.3. Effect on the relative weight of the analyzed organs The ROW of both kidneys, liver and spleen in the untreated group has increased when compared to the normal control group (p < 0.01, p < 0.001, p < 0.05 respectively). The administration of APA 500 mg/kg b.w. to normal animal (group III) decreased the relative weight of kidneys (p < 0.05) (Table 4).

Table 1 Effect of the two extracts of Paronychia argentea on the relative body weight (RBW) of Wistar rats. Relative body weight, RBW (%)

Day 7

Normal group control Lithiasic group control Normal group treated (500 mg/kg) APA APA (250 mg/kg) APA (500 mg/kg) BPA (10 mg/kg) BPA (20 mg/kg) Normal group treated (20 mg/kg) BPA

23.78 −5.72 13.71 14.75 6.02 −0.22 5.82 7.55

Day 14 ± ± ± ± ± ± ± ±

2.02 3.86a , *** 2.47a , * , b , ** 2.87a , * , b , ** 1.81a , *** , b , * 4.12a , *** , b , * 4.33a , ** 4.74a , *

33.39 −0.35 14.73 29.66 11.51 12.27 17.02 19.49

± ± ± ± ± ± ± ±

Day 21 3.60 2.07a , *** 1.31a , ** , b , *** 6.08b , ** 3.07a , ** , b , * 3.52a , ** , b , * 5.14a , * , b , * 5.41b , **

50.39 7.52 24.88 43.12 15.27 23.24 27.43 27.20

± ± ± ± ± ± ± ±

Day 28 3.30** 2.32a , *** 0.72a , ** , b , ** 5.06b , *** 4.22a , *** 4.23a , ** , b , * 6.39a , * , b , * 10.10

65.55 10.39 35.01 56.42 30.95 33.52 37.92 43.88

± ± ± ± ± ± ± ±

3.98*** 2.21a , *** 1.41a , ** , b , *** 4.72b , *** 4.96a , *** , b , ** 4.51a , ** , b , * 5.93a , ** , b , ** 5.91a , * , b , **

APA, aqueous extract of Paronychia argentea; BPA, butanolic extract of Paronychia argentea. The values are expressed as mean ± S.E.M. (n = 6 animals/group). Analyzed by Student test (impaired). a Comparisons are made with Group I. b Comparisons are made with Group II. * Statistically significant at p < 0.05. ** Statistically significant at p < 0.01. *** Statistically significant at p < 0.001.

Table 2 Effect of the two extracts of Paronychia argentea on the biochemical and haematological parameters in Wistar rats treated for 28 consecutive days. Lithiasic group control (II)

Normal group control (I) Urea (g/dL) Creatinine (mg/L) Uric acid (mg/L) Calcium (mmol/L) Potassium (M. Eq/L) Sodium (M. Eq/L) Magnesium (mmol/L) Proteins AST (U/L) ALT (U/L) PAL (UI/L)

0.35 7.45 11.25 2.70 8.13 138.20 1.31 73.72 135.3 52.07 283.50

± ± ± ± ± ± ± ± ± ± ±

0.06 0.71 1.49 0.49 0.25 0.48 0.06 1.13 0.81 4.49 0.06

0.55 9.61 12.71 2.34 6.88 140.33 1.52 81.82 151.72 66.88 401.3

± ± ± ± ± ± ± ± ± ± ±

Normal group treated (500 mg/kg) (III)

0.06a , * 0.51a , * 1.32 0.44 0.31a , ** 1.41 0.16 1.09 7.27 2.12a , * 17.50a , **

0.35 7.43 9.64 2.78 6.62 137.82 1.22 71.5 134.75 55.00 278.70

± ± ± ± ± ± ± ± ± ± ±

Normal group treated (20 mg/kg) BPA (IV)

0.01b , ** 0.12b , ** 0.75 0.06 0.28a , ** 0.48 0.05 1.15b , *** 0.86 3.66b , * 11.10

0.35 8.08 11.09 2.82 6.42 139 1.37 79.79 134.8 52.73 271.80

± ± ± ± ± ± ± ± ± ± ±

APA (250 mg/kg) (V)

0.01b , ** 0.30b , * 0.94 0.08 0.36a , ** 0.58 0.09 1.92 4.76 1.81b , * 16.50

0.41 8.50 13.20 2.43 7.60 137.83 1.74 72.89 148.68 59.98 371.50

± ± ± ± ± ± ± ± ± ± ±

0.04 0.20 1.76 0.23 0.25 0.65 0.24 1.51 4.75 2.38b , * 17.80a , **

APA (500 mg/kg) (VI) 0.35 7.17 11.74 2.48 7.26 138.88 1.52 80.95 148.00 66.15 428.00

± ± ± ± ± ± ± ± ± ± ±

0.02b , ** 0.93 1.19 0.15 0.37 0.41 0.12 2.06a , * 4.75 3.13a , * 25.10a , **

BPA (10 mg/kg) (VII) 0.32 8.57 13,87 2.75 6.50 137.67 1.12 71.89 141.32 43.92 354.80

± ± ± ± ± ± ± ± ± ± ±

0.05b , ** 0.20 1.02 0.09 0.25a , *** 1.02 0.14 1.09b , ** 9.73 1.37b , *** 10.20a , * , b , *

BPA (20 mg/kg) (VIII) 0.35 8.42 13.02 2.79 6.50 140.00 1.40 76.92 139.05 44.92 332.30

± ± ± ± ± ± ± ± ± ± ±

0.02b , ** 0.25 0.94 0.08 0.25a , *** 0.58 0.09 1.09 2.56 1.12b , *** 28.50

Table 3 Effect of the two extracts of Paronychia argentea on the haematological parameters in Wistar rats treated for 28 consecutive days. Normal group control WBC (×10−3 /␮L) RBC (×10−6 /␮L) Haemoglobin (g/dL) Hematocrit (%) MCV (fl) MCH (pg) MCHC (g/dL) Platelets (×10−3 /␮L) Lymphocytes (%) Lymphocytes (×10−3 /␮L)

10.32 6.84 12.96 40.02 58.50 18.92 32.6 953.20 82.66 8.36

± ± ± ± ± ± ± ± ± ±

1.47 0.06 0.21 0.79 0.88 0.88 0.10b , * 51.50 3.91 1.04

Lithiasic group control 8.28 7.66 14.13 49.42 64.62 18.48 28.65 1003.70 78.05 6.38

± ± ± ± ± ± ± ± ± ±

0.93a , * 0.08a , *** 0.08a , *** 0.36a , ** 0.87a , *** 0.14 0.26a , *** 70.10 3.08 0.62

Normal group treated (500 mg/kg) 10.15 7.39 13.15 41.36 60.38 19.15 31.73 950.00 87.42 8.85

± ± ± ± ± ± ± ± ± ±

1.13 0.57 0.32 1.80b , ** 0.98b , ** 0.40 0.67b , *** 45.30 3.13 0.01

Normal group treated (20 mg/kg) BPA 10.55 7.37 13.59 40.58 60.09 18.73 30.59 1044.50 87.38 9.05

± ± ± ± ± ± ± ± ± ±

0.50 0.20 0.09a , * , b , ** 0.10b , *** 0.55b , ** 0.23 0.30b , *** 64.40 2.87 0.54

APA (250 mg/kg) 10.19 7.30 13.76 47.69 65.27 18.86 28.89 933.70 85.71 8.45

± ± ± ± ± ± ± ± ± ±

0.61b , ** 0.11a , ** , b , * 0.15a , ** , b , * 0.84a , *** 0.79a , *** 0.12 0.30a , *** 37.40 1.88 0.56

APA (500 mg/kg) 9.70 7.51 13.70 47.99 63.98 18.25 28.58 918.80 82.82 7.90

± ± ± ± ± ± ± ± ± ±

0.86 0.18a , * 0.20a , * 1.01a , *** 0.85a , *** 0.26 0.22a , *** 37.40 1.82 0.71

BPA (10 mg/kg) 8.62 6.54 12.45 40.06 61.48 19.00 31.05 896.10 83.70 8.38

± ± ± ± ± ± ± ± ± ±

0.64 0.25b , ** 0.43b , ** 1.41b , *** 1.64b , *** 0.12 0.88 91.50 3.69 0.67

BPA (20 mg/kg) 9.55 6.98 13.02 44.79 64.09 18.65 29.11 1034.50 88.64 8.38

± ± ± ± ± ± ± ± ± ±

0.50 0.15b , ** 0.20b , *** 1.04a , ** , b , ** 0.58a , *** 0.19 0.24 56.60 2.67 0.51

S. Bouanani et al. / Journal of Ethnopharmacology 129 (2010) 38–45

APA, aqueous extract of Paronychia argentea; BPA, butanolic extract of Paronychia argentea. The values are expressed as mean ± S.E.M. (n = 6 animals/group). Analyzed by ANOVA followed by Dunnett’s multiple comparison test. AST, aspartate aminotransferase; ALT, alanine aminotransferase; PAL, alkaline phosphatase. a Comparisons are made with Group I. b Comparisons are made with Group II. * Statistically significant at p < 0.05. ** Statistically significant at p < 0.01. *** Statistically significant at p < 0.001.

APA, aqueous extract of Paronychia argentea; BPA, butanolic extract of Paronychia argentea. The values are expressed as mean ± S.E.M. (n = 6 animals/group). Analyzed by ANOVA followed by Dunnett’s multiple comparison test. WBC, white blood cell; RBC, red blood cell; MCV, mean corpuscular volume; MCH, mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin concentration. a Comparisons are made with Group I. b Comparisons are made with Group II. * Statistically significant at p < 0.05. ** Statistically significant at p < 0.01. *** Statistically significant at p < 0.001.

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Table 4 Effect of the two extracts of Paronychia argentea on the relative body weights of kidney, liver and spleen in Wister rats treated for 28 consecutive days. Groups

Body weight (g)

Normal group control (I) Lithiasic group control (II) Normal group treated (500 mg/kg) (III) Normal group treated (20 mg/kg) BPA (IV) APA (250 mg/kg) (V) APA (500 mg/kg) (VI) BPA (10 mg/kg) (VII) BPA (20 mg/kg) (VIII)

189.67 139.08 184.16 187.46 151.02 159.90 158.65 159.47

Relative body weight (%) Kidney 0.60 0.75 0.47 0.59 0.59 0.61 0.64 0.62

± ± ± ± ± ± ± ±

Liver 0.02 0.02a , ** 0.05a , * 0.01b , *** 0.01b , *** 0.02b , ** 0.02b , * 0.01b , **

3.29 4.38 3.47 3.47 4.07 4.06 3.84 3.87

Spleen ± ± ± ± ± ± ± ±

0.06 0.23a , *** 0.15b , ** 0.08b , ** 0.20a , ** 0.15a , ** 0.17a , * , b , * 0.07a , * , b , *

0.21 0.17 0.24 0.21 0.20 0.19 0.21 0.23

± ± ± ± ± ± ± ±

0.06 0.02b , * 0.02a , * 0.01 0.01 0.01b , * 0.01 0.02

APA, aqueous extract of Paronychia argentea; BPA, butanolic extract of Paronychia argentea. The values are expressed as mean ± S.E.M. (n = 6 animals/group). Analyzed by ANOVA followed by Dunnett’s multiple comparison test. a Comparisons are made with Group I. b Comparisons are made with Group II. * Statistically significant at p < 0.05. ** Statistically significant at p < 0.01. *** Statistically significant at p < 0.001.

All doses of the two extracts produced a significant decrease in the relative weight of kidneys to normal values, whereas only the administration of the butanolic extracts of PA re-established the liver weight to normal. There was no significant change on the relative body weight of spleen in all the treated groups. 3.2.4. Histopathology finding No alteration was observed in the organs of the normal animals as well in the normal animals treated with 500 mg/kg (group III) and 20 mg/kg (group IV). Figs. 1 and 2 show photomicrography of the kidney and liver respectively and Table 5 summarizes the histopathological features observed in all animals.

Liver: histopathological features of the liver of control rats showed normal structures with well presented cytoplasm (CP), well brought and central vein (NC), prominent nucleus and nucleolus (Fig. 2). The administration of 500 mg of the aqueous extract induced steatosis and hemosederin deposits in few parts of liver (in two animals). Kidney: histopathological features of the kidneys of control rats showed normal features with prominent cortical tubules and Bowman’s capsule (BC) (Fig. 1). The examination of the paraffin kidney sections revealed that in all the groups treated, no crystal was found when compared to untreated group which revealed an important necrosis and a few basophile deposits. We noted a necrosis in only a few part of the kidney (+) and there were none in four animals (−) in the groups V (APA 250 mg/kg b.w.)

Fig. 1. Paraffin section viewed under polarized light of (A) a control rat kidney ‘control group’ showing normal tubular brush-border and intact glomeruli (GL) and Bowman’s capsule, (B and C) a kidney from a rat that received distilled water only ‘untreated group II’ where numerous crystals in the cortex, tubular and medulla can be seen, and (D) a kidney from a rat that received PA at 500 mg/kg of APA showing moderate necrosis (400× magnification).

S. Bouanani et al. / Journal of Ethnopharmacology 129 (2010) 38–45

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Fig. 2. Paraffin section viewed under polarized light of (A) a control rat liver ‘control group’ showing normal hepatic cells with well presented cytoplasm, well brought and central vein (NC), prominent nucleus and nucleolus, (B) a liver from a rat from ‘untreated group’ where massive fatty changes (steatosis) can be seen, and (C) a liver from a rat that received 500 mg/kg of aqueous extracts of Paronychia argentea ‘treated group’ showing well brought out central vein and few hemosederin (H) deposits.

Table 5 Effect of the two extracts of Paronychia argentea on the kidney, liver histopathology in Wistar rats treated for 28 consecutive days. Groups

Degree of kidney injury

Degree of liver injury

Kidney necrosis

Normal group control (I) Lithiasic group control (II) Normal group treated (500 mg/kg) (III) Normal group treated (20 mg/kg) BPA (IV) APA (250 mg/kg) (V) APA (500 mg/kg) (VI) BPA (10 mg/kg) (VII) BPA (20 mg/kg) (VIII)

Lymphocyte infiltration

0

+

++

+++

6 0 6 5 0 0 0 0

0 1 0 1 3 4 4 3

0 2 0 0 1 1 2 2

0 3 0 0 1 1 0 1

0 1/6 0 0 0 2/6 0 0

Steatosis

Hemosederin deposits

0

1

2

3

6 2 6 6 5 5 6 6

0 2 0 0 1 1 0 0

0 0 0 0 0 0 0 0

0 2 0 0 0 0 0 0

0 4/6 0 0 1/6 0 0 0

APA, aqueous extract of Paronychia argentea; BPA, butanolic extract of Paronychia argentea. −, 0 (normal); +, 1 (little effect); ++, 2 (appreciable effect); +++, 3 (severe effect). Little effect: tubular necrosis in few number of tubule with glomerular congestion and oedema; appreciable effect: necrosis in appreciable number of tubule; severe effect: massive necrosis with hemorrhage in an important number of tubule.

and VII (BPA 10 mg/kg b.w.). However, only two animals exhibited a normal feature (−) in the higher dose of the aqueous extract. Four kidneys of the group treated with the higher dose of the butanolic extract revealed little necrosis and moderate in two animals (++). PA treatment was found to have no effect on the tissue section of the spleen (data not shown). 4. Discussion In general, traditional medicine uses plants without taking into account the toxicity aspect. Our attention was particularly focused on phytotherapy, which is common in traditional medicine as an alternative to primary healthcare in many countries. Accordingly, the aerial parts of Paronychia argentea, a plant widely distributed in the Mediterranean area, are used in traditional medicine in Algeria to treat kidney stones (Beloued, 1998). From the testimony of herbalists and patients with lithiasis, this plant is widely known for

its ability to aid in dissolving and expelling stones from the urinary tract after a few days of treatment (Laroubi et al., 2007). However, pharmacological tests or toxicity studies about this plant are rare. In the present study, we induced calcium oxalate nephrolithiasis in rats. Then, we treated these rats with plant extracts in order to test its ability to dissolve nephrolithiasis described in traditional medicine. Evidence in previous studies indicated that 6 h after injection of sodium oxalate in rats, crystals of CaOx were present mainly as large aggregates near the papillary tip with increased tubular necrosis. Loss of tubular epithelium was more pronounced and there were more cellular fragments in the tubular lumens (Khan et al., 1982). The biochemical mechanisms for this process are related to an increase in the urinary concentration of oxalate. Similar results have been obtained when rats were treated with ethylene glycol and with ethylene glycol and ammonium oxalate (Adhirai and Selvam, 1998). In the present work, acute and sub acute toxicity assays have been carried out in normal and calcium oxalate nephrolithiasis

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model. Preliminary acute toxicity has shown that the LD50 of the aqueous and the butanolic extracts were respectively higher than 2000 and 40 mg/kg. Loss weight observed in untreated group is due to anorexia due to disturbances in carbohydrates, proteins or fat metabolism which is affected by the injection of sodium oxalate. Indeed the toxic nature of oxalic acid and its nephrotoxicity was well recognized in the nineteenth century (Hodgkinsons, 1977). Its injurious effects are considered to be a result of the physical properties of its calcium salts (calcium oxalates) which are insoluble at physiological pH inducing calcium oxalate (CaOx) nephrolithiasis. However other study indicated that free oxalate levels upper 140 ␮M led to increased DNA fragmentation, membrane permeability and produced a number of morphological changes including cytoplasmic vacuolization and nuclear pyknosis (Thamilselvan and Khan, 1998). This injury may be exaggerated in the presence of CaOx crystals (Khan et al., 1982). When compared to untreated group, all tested doses produced a gain in the body weights but it was still less than the observed gain in normal group, in the exception of the animals treated with 500 mg/kg b.w. of APA (group III). This observation may be due to the fact that this concentration of aqueous extract stimulates the absorption or metabolism of nutrients. The drugs inducing nephrotoxicities are often associated with marked elevations in blood urea and acute tubular necrosis (Adeneye and Adokiye, 2008). In urolithiasis, the glomerular filtration rat decreases due to the obstruction of the outflow of urine by stones in urinary system. Due to this, the waste products particularly nitrogenous substances such as proteins, urea, creatinine and uric acid accumulate in blood (Grover and Resnick, 1995). In this study, intraperitoneal injection of sodium oxalate induced nephrotoxicities. These toxicities were characterized by marked elevation of blood urea and serum creatinine. While uric acid remained normal, a remarkable decrease to normal value in renal urea and creatinine levels was observed in the groups treated with BPA and APA 500 mg/kg b.w. compared to the untreated group which showed an elevation in these two parameters. This view is also strengthened by the fact that the relative weights of the kidneys in this group show evidence of toxicity. However, these elevations were attenuated by treatment with the two extracts. In accordance with the several finding of several authors, alteration in the structure of renal tubules was limited to their crystal containing portions. They were dilated and showed necrosis of their lining epithelia, whereas neighbouring tubules without crystal appeared normal. The intensity of necrosis, the size of CaOx particles, their number and their distribution within the inner medulla appeared to be dependent on the time interval following NaOx injection (Khan et al., 1982). The degrees of tubular necrosis were increased in the untreated group (++), all the animal showed extensive necrosis. The kidney histology revealed a characteristic effect ranging from vascular congestion or haemorrhage to diffuse cortical necrosis, confirming the observed changes in renal creatinine and urea levels. Whereas in all the treated groups, except the two control treated groups, a little and moderate necrosis was also observed in the histopathological sections of the kidneys in the majority of animals, indicating a reduction of the extent of damage done at the tissue level (Wiessner et al., 2001). It suggests that the effect of the extracts could be advantageous in preventing urinary stone retention by reducing renal necrosis and thus inhibit crystal retention. Previous studies indicate that both individual cell injury (loss of lipid asymmetry) and generalized cell monolayer injury (loss of cell polarity) result in the presentation of different cell surface, and that both form of injury result in an increased affinity of crystal attachment (Wiessner et al., 2001). In addition, the significant decreases of the kidney index in the normal animals treated with aqueous extract were not associated

with any histopathological changes. Indeed, the aqueous extract was not nephrotoxic at this concentration. The observed necrosis in the higher dose of that extract in the lithiasic groups was attributed to the nephrotoxicity of oxalate. In our study, marked proteinimia was observed in animals of group II. The restoration to normal levels after treatment with aqueous extract of PA at the dose of 250 mg/kg and BPA extracts shows that these doses help in minimizing the extent of tubular dysfunction. Hepatic function has been monitored by the evaluation of the serum level of ALT, AST and PAL. The AST and ALT activities are known as cytosolic marker enzymes reflecting hepatocellular necrosis as they are released into the blood after cell membrane damage (Latha et al., 1998). In the present study, both enzyme activities were used as indicator of hepatic damage. Compared with normal rats, lithiasis rats showed more activities of serum AST and ALT. There was a significant difference between the two extracts. The significant elevation in the ALT levels with high doses of treatments tested in this study (500 mg/kg) may be an indicator of hepatocellular damage (Latha et al., 1998). Body cells contain more AST than ALT. Usually, about 80% of AST is found in the mitochondria whereas ALT is a purely cytosolic enzyme. The AST is also found in a large number of tissues, such as heart, lung, skeletal muscle and kidney, whereas ALT is primarily limited to liver. Thus the latter is considered as highly sensitive indicator of hepatotoxicity (Al-Mamary et al., 2002). The ALT in blood increases when the hepatic cellular permeability is changed or when necrosis and cellular injury occur. A rise in plasma alkaline phosphatase (PAL) levels is usually a characteristic finding in obstructive hepatobiliary disease as found in cholestasis liver disease (Al-Mamary et al., 2002). To confirm this assertion, the liver histology reveals evidences of steatosis and hemosederin deposits at this dose (APA 500 mg/kg b.w.). The reactive oxygen species may be involved in the development of steatohepatitis (Garcia-Ruiz et al., 1995). In this view, the reduction of AST and ALT levels with the butanolic extract is a stabilization indication of plasma membrane as well as repair of hepatic tissue damage caused by oxalate crystal deposition. The haematological tests revealed a significant increase in the red blood cells in the groups treated by the aqueous extract (p < 0.01). This implies that there may be possible increase in erythropoiesis increasing doses of the aqueous extracts when it is associated with elevation in the packed cell volume and haemoglobin level (Muibat et al., 2007). No significant change in the lymphocyte levels could be an attestation of the fact that PA extract at the tested doses may not contain biologically active principle that have the ability to boost the immune system through increasing the population of defence white blood cells. The spleen after treatment with all doses of PA was not affected judging by the absence of significant change in its relative weight or histopathological damage. This shows that there is not any possible immunotoxic effect of PA at the tested doses since the spleen plays a major role in immunological mechanisms. Several studies have shown that cellular membrane, damaged proximal tubules or loop of Henle, injured urothelium after treatment with nephrotoxic agents such as gentamicin, induce nucleation of CaOx crystals at a lower supersaturation (Adhirai and Selvam, 1998). Membrane damage due to lipid peroxidation or depletion of cellular antioxidant have been suggested as a predisposing factor for CaOx crystals deposition (Selvam and Bijikurien, 1991). Recent study show that the butanolic extract of Paronychia argentea can prevent or slow down the oxidative damage induced by chloropyrifos a chemical pesticide (Zama et al., 2007). This extract may play a crucial role to prevent both lipid peroxidation and crystal aggregation. Our current data suggest a mechanism where PA extract used in traditional medicine might prevent and

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possibly eliminate pre-existing kidney stones. It is interesting to analyze the results in view of the chemical composition of PA. In a recent study, PA butanolic extract was shown to contain a new flavonol glycoside and two oleanane saponins (Braca et al., 2008). Saponin derivatives appear as component of a great number of medicinal herbs with claimed antiurolithiasis properties (Lakshminarasimhan et al., 2002). 5. Conclusion In conclusion, the presented data indicate that administration of the butanolic PA extract of aerial parts to rats with NaOx induced lithiasis, reduced and prevented the growth of urinary stones, supporting folk information regarding antiurolithiatic activity of the plant. The mechanism underlying this effect and the active ingredients which differentiate the two extracts is still unknown. Apparently, it could be related to lowering of urinary concentrations of stone forming constituents, antioxidant activity and free radical scavenging principle(s) contained in the extract. These effects could confirm the antiurolithiatic property of PA extracts. The acute and sub acute oral administration of Paronychia argentea extracts did not induce significant alterations in almost all biochemical, haematological and morphological parameters in Wistar rats. This investigation could be regarded as preliminary probes, requiring further studies to establish the mechanism of toxicity or pharmacological effects. Prospective studies should include among other investigations. Acknowledgements We thank Dr. O. Laouar, from the Anatomopathology Department of CHU IBEN ROCHED (Annaba, Algeria), for helping us to carry out histopathological studies. We thank Dr. G. Saladin too from the Limoges University (France) for the identification of the plant. References Adeneye, A.A., Adokiye, S.B., 2008. Protective effect of the aqueous leaf and seed extract of Phyllanthus amarus on gentamicin and acetaminophen-induced nephrotoxic rats. Journal of Ethnopharmacology 118, 318–332. Adhirai, M., Selvam, R., 1998. Renal calcium oxalate binding protein: studies on its properties. Kidney International 53, 125–129. Afifi, F.U., Al-Khalidi, B., Khalil, E., 2005. Studies on the in vivo hypoglycemic activities of two medicinal plants used in the treatment of diabetes in Jordanian traditional medicine following intranasal administration. Journal of Ethnopharmacology 100, 314–318. Al-Bakri, A.G., Affifi, F.U., 2007. Evaluation of antimicrobial activity of selected plant extracts by rapid XTT colorimetry and bacterial enumeration. Journal of Microbiological Methods 68, 19–25. Al-Mamary, M., Al-Habori, A.M., Al-Aghbari, M., Baker, M., 2002. Investigation into the toxicological effects of Catha edulis leaves: a short term study in animals. Phytotherapy Research 16, 127–132. Araújo Viel, T., Diogo Domingos, C., da Silva Monteiro, A.P., Riggio Lima-Landman, M.T., Lapa, A.J., Souccar, C., 1999. Evaluation of the antiurolithiatic activity of the extract of Costus spiralis Roscoe in rats. Journal of Ethnopharmacology 66, 193–198.

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