Phytomedicine 21 (2014) 523–528
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Diuretic and antilithiasic activities of ethanolic extract from Piper amalago (Piperaceae) Antônio da Silva Novaes a , Jonas da Silva Mota b , Andersson Barison c , Clebson Luiz Veber c , Fábio Juliano Negrão a , Candida Aparecida Leite Kassuya a , Márcio Eduardo de Barros a,d,∗ a
Department of Health Sciences, Federal University of Grande Dourados, MS, Brazil Department of Biodiversity Research, University of the State of Mato Grosso do Sul, Dourados, MS, Brazil c Department of Chemistry, Polytechnic Center, Federal University of Paraná Federal University, Curitiba, PR, Brazil d General Hospital, Federal University of Grande Dourados, MS, Brazil b
a r t i c l e
i n f o
Article history: Received 4 July 2013 Received in revised form 4 September 2013 Accepted 6 October 2013 Keywords: Piper amalago Diuretic activity Urolithiasic Piperaceae
a b s t r a c t Objective: Piper amalago is used in Brazilian folk medicine as diuretic and for the treatment of urinary calculus disease, although no scientific data have been described to support these effects. Thus, this study aims to evaluate the diuretic effects and antilithiatic activity of the ethanolic extract of P. amalago (EEPam). Materials and methods: Ethanolic extracts of P. amalago (125, 250 and 500 mg/kg) were orally administered in male Wistar rats (n = 5) and urinary excretion was measured at intervals of up to 24 h after administration. The antilithiasic effect of EEPam on calcium oxalate urolithiasis crystallization was examined in a turbidimetric model. Results: The oral administration of all doses of EEPam significantly increased urine output after 24 h when compared to control group. Moreover, the application of EEPam, induced an inhibitory effect on calcium oxalate crystallization. Conclusions: According to results, P. amalago extracts showed diuretic and natriuretic activity and antilithiasic effects. © 2013 Elsevier GmbH. All rights reserved.
Introduction Diuretic drugs are clinically used to the treatment of oedema, hypertension, urolithiasis and others diseases (Orjala et al., 1992; Pietrow and Karellas, 2006; Rose, 1991; Wright et al., 2007). Urolithiasis is a disturbance that affects kidney, which is considered a systemic disorder associated with bone loss and fractures, chronic kidney disease, hypertension, increased risk of coronary artery disease, metabolic syndrome, and type-2 diabetes mellitus (Sakhaee et al., 2012). In addition, diuretics drugs are also used in the treatment of these diseases (Carter, 2012). However, diuretic drugs produce several adverse effects such as diabetes, hypokalemia and others (Zillich et al., 2006) while antilithiasic drugs have only preventive and protective effect (Allie-Hamdulay and Rodgers, 2005; Erickson et al., 2011; Tseng and Preminger, 2011; Xu et al., 2011). In this context, the search for new agents with diuretic and antilithiasic properties is of interest for public health.
∗ Corresponding author at: Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Dourados/MS 79825-070, MS, Brazil. Tel.: +55 67 3410 2321; fax: +55 67 3410 2326. E-mail addresses: [email protected], [email protected] (M.E. de Barros). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.10.014
In Brazil, there are several plants that are frequently used in folk medicine as diuretic drugs such as Piper amalago. This plant is also used against cardiovascular problems like hypertension and renal disturbances like renal stones (Achenbach et al., 1986; Coimbra, 1994; Dias et al., 1984; Parmar et al., 1997). In addition, traditional use of P. amalago commonly known in Brazil as “jaborandi” is used to alleviate chest pain and inflammation (Achenbach et al., 1986; Domínguez and Alcorn, 1985; Domínguez et al., 1986; Parmar et al., 1997). Besides, the tea from the leaves of P. amalago is used in treatment of burns (Alves et al., 2008). A neurobehavioral study showed that the methanolic extract from leaves of P. amalago acts on the central nervous system, with anxiogenic properties in rats evaluated in the model of the elevated plus-maze model affecting locomotion and exploration in this model, but without inducing genetic toxicity (Lopes et al., 2012). In the state Mato Grosso do Sul, Brazil, Piper species are used for several disorders (Alves et al., 2008). An ethnobotanical survey in Dourados, MS, Brazil, found that of the families identified, 10.8% were from Piper species, showing a large amount of species from this genus in the region (Alves et al., 2008). Phytochemical investigations showed that plants of the family Piperaceae have several compounds such as esters, phenolic ethers, volatile oils, lignans, and pyrrolidine alkaloids
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(Gibbs, 1974). Regarding the genus Piper L. several phytochemical groups such as lignans, flavonoids, lactones, alkaloids (pyrrolidines and piperidines), butenolides and cyclohexane epoxide have been found (Calle-Alvarez, 1983; Nair and Burke, 1990; Sengupta and Ray, 1987). Considering that the drugs currently available for the treatment of renal disturbances show undesirable side effects and to the best of our knowledge, no scientific data about the activity of P. amalago in the renal system have been described. The aim of this study was to assess diuretic, natriuretic and kaliuretic activities of these medicinal plants. Furthermore, in vitro antilithiasic activity was also investigated. Materials and methods Plant material Leaves of P. amalago were collected in Dourados, MS, Brazil in August, 2008 and identified by Prof. Elsie Franklin Guimarães from Jardim Botânico Research Institute of Rio de Janeiro, RJ, Brazil. A voucher specimen (DDMS 4410), was deposited in the herbarium of the Federal University of Grande Dourados, MS, Brazil. Ethanolic extract and phytochemical analysis Dried at room temperature and powdered leaves of P. amalago (816 g) was extracted with ethanol 92% for 7 days by maceration. The solvent was then eliminated under reduced pressure to 1/6 of initial volume and then freeze-dried, yielding 4.2% (w/w). P. amalago ethanolic extract (30 g) was submitted to column chromatography over silica gel eluted with increasing concentrations of hexane in AcOEt. The chromatographic fractioning resulted in 1 (32.0 mg), 2 (11.5 mg), 3 (5.1 mg) and 4 (5 mg) (Fig. 1). The compounds were identified by exhaustive analyses of 1D and 2D NMR data and compared with literature data (Alécio et al., 1998; Carrara et al., 2012; Domínguez et al., 1985, 1986; Jacobs et al., 1999; Nascimento et al., 2012). During the experiments, the freeze-dried extract was dissolved in distilled water with Tween 80. Chemicals and general procedures Furosemide from Lasix, Sanofi-Aventis Farmacêutica, a highceiling loop diuretic, was used as reference drug (positive control). It was dissolved in distilled water prior to administration. EDTA, Tween 80, Sodium oxalate and Fluid Pack were of analytical grade. NMR data were acquired on a Bruker AVANCE III 400 NMR spectrometer operating at 9.4 Tesla, observing 1 H and 13 C nuclei at 400 and 100 MHz, respectively. Animals The experiments were performed with male adult 8-weekold Wistar rats provided by the vivarium of Federal University of Grande Dourados. The animals were maintained under a 12 h light–dark cycle, 60–80% humidity at 22 ± 1 ◦ C and received food and water ad libitum. Rats were handled in accordance with internationally accepted standard guidelines for use of animals. All procedures were approved by the Ethics Committee for research on humans and animals, under number 034/2011 and 011/2011, respectively. Diuretic activity assay Diuretic activity was determined according to Kau et al. (1984) method (Kau et al., 1984), with some modifications. Brief, rats after
Fig. 1. Chemical compounds found in leaves of Piper amalago (Piperaceae).
overnight fasting and water ad libitum were divided into five groups (n = 5). Before treatment, all animals received physiological saline (0.9% NaCl) at an oral dose of 5% body weight to impose a uniform water and salt load (Benjumea et al., 2005). Then, ethanolic extract forms P. amalago were orally administered to the animals at doses of 125, 250 and 500 mg/kg (b.w.). Negative control group received the same amount of distilled water while those from positive control received 10 mg/kg (b.w.) of furosemide. Immediately after administration, the animals were placed in metabolic cages. Urine was collected and measured at 1, 2, 4, 6, and 24 h. Cumulative urine excretion was calculated in relation to body weight and expressed as ml/100 g b.w. Electrolyte (Na+ , K+ ) concentrations were estimated from each urine sample of each rat at all time intervals using Roche 9180 Electrolyte Analyzer and expressed as mmol/100 g (b.w.) and pH was verified at the end of experiments, as well. The diuretic index (volume treated group/volume control group and saluretic (mmol/l treated group/mmol/l control group) was calculated.
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Table 1 Effect of single oral EEPam administration on urine volume and electrolyte excretion. Treatment
Vehicle Furosemide EEPam EEPam EEPam
Dose (mg/kg)
– 10 125 250 500
Urine volume (ml/100 g/24 h)
4.1 6.8 6.36 5.51 5.44
± ± ± ± ±
0.3 0.2*** 0.2*** 0.2* 0.4*
Diuretic indexa
– 1.67 1.54 1.34 1.32
Na+ (mmol/l/100 g/24 h)
477 682 660 590 667
± ± ± ± ±
10.1 23.8*** 14.1*** 37.9** 38.6***
K+ (mmol/l/100 g/24 h)
428 548 520 524 537
± ± ± ± ±
15.2 21.8*** 7.0*** 16.3*** 17.7***
Saluretic indexb Na+
K+
– 1.42 1.38 1.23 1.39
– 1.28 1.21 1.22 1.25
Data are expressed as mean ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001 in comparison with the control group (vehicle) using One-Way variance test (ANOVA) followed by Student–Newman–Keuls test. a Diuretic index = volume treated group/volume control group. b Saluretic index = mmol/l treated group/mmol/l control group.
Blood serum analysis After diuretic experiments, the animals were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) and blood samples (3–5 ml) collected in tubes containing EDTA (1 mg/ml) by puncture from renal vein. Plasma was obtained by 10 min centrifugation at 4000 rpm. The plasma was separated and stored at −20 ◦ C for further analysis. Na+ and K+ levels in plasma were determined by an automatic Roche, 9180 Eletrolyte Analyzer.
test, or Student’s t-test. P-values less than 0.05 were considered statistically significant. Graphs were drawn and statistical analysis was carried out using the GraphPad Prism software version 5.00 for Windows (GraphPad Software).
Antilithiasic activity
The phytochemical analysis of ethanolic extract from the leaves of P. amalago showed the presence of abundant pyrrolidide amides as well as chalcones and a flavonol (Fig. 1).
Calcium oxalate crystallization was induced in the urine obtained from healthy individuals. Human urine samples were obtained from six healthy subjects, three men and three women, with no personal or family history of kidney stone disease. Human urine samples were centrifuged at 5000 rpm for 8 min, the supernatant was then transferred to a clean tube, the pH was adjusted to 6.0 and it was filtered through a 0.22 m filter (Barros et al., 2003). The in vitro calcium oxalate crystallization protocol was modified, according with some methods described in literature (Atmani and Khan, 2000; Atmani et al., 1996; Balaji and Menon, 1997; Barros et al., 2003; Dussol and Berland, 1998; Robertson et al., 1968). Calcium oxalate precipitation was induced by adding 50 l of 0.1 M sodium oxalate per ml of urine (corresponding to 6.7 mg/ml), every 15 min (0, 15, 30 and 45 min) under shaking at 37 ◦ C, resulting in final urine concentration of 26.8 mg/ml. Each urine sample was divided into two aliquots, one of which was used as control (crystallization without Piper extract) while the other CaOx precipitation was induced in the presence of Piper extract, which was added to the sample 30 min before the crystallization process. Freezedried Piper amalago extract was resuspended in distilled water, filtered through 0.22 m filter, and used at final urine concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 2.5 and 5 mg/ml, based on a concentration-response curve. The crystals obtained were semiquantitative estimated by turbidity, immediately after crystallization. Aliquots of 100 l were loaded onto a 96 well microplate and the absorbance was measured on a plate reader (Thermoplate Reader), at 630 nm in quadruplicate and the average was used to calculate the turbidity index TI = (DOt × DOb)/DOb, where DOt is the mean sample absorbance after calcium oxalate precipitation and DOb is the mean sample absorbance before precipitation (Barros et al., 2003). The number and size of crystals were determined with aid of a Neubauer counting chamber. Statistical analysis The results are expressed as mean ± S.E.M. of experiments. Statistical significance was determined through one-way analysis of variance (ANOVA), followed by either Newman–Keuls test, Dunnet
Results Phytochemical analysis
Diuretic activity The effects of the oral administration of ethanolic extract of P. amalago on the urinary outputs are shown in Fig. 2 and Table 1. A significant increasing in urinary excretion (p < 0.001) was observed for 125 mg/kg of EEPam for 1, 2, and 24 h (p < 0.001), as well as for 500 and 250 mg/kg (p < 0.05) (Fig. 2). The effect of a single dose of reference diuretic, furosemide, induced a significant increase in the Na+ excretion at all times analyzed during the 24 h of experiment (P < 0.001). The amount of urinary electrolytes (Na+ and K+ ) was measured in all samples collected at 1, 2, 4, 6 and 24 h as shown in Figs. 3 and 4. The positive
Fig. 2. Time course of urinary volume excretion in rats treated with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. The urine volume was measured at 1, 2, 4, 6 and 24 h after treatment. Cumulative values are reported as mean ± SEM for five rats in each group. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig. 3. Effect of single oral administration with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. Urine samples were collected at 1, 2, 4, 6 and 24 h after treatment and sodium levels were analyzed. Each bar represents the mean of five animals, and vertical lines show the S.E.M. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) (*p < 0.05; **p < 0.01; ***p < 0.001).
control group revealed that the amount of Na+ and K+ significantly increased in all times evaluated. The EEPam dose of 125 mg/kg was the most potent, with a diuretic index of 1.54. Equally important, for doses of 250 and 500 mg/kg, the diuretic indexes were 1.34 and 1.32, respectively (Table 1). Na+ and K+ levels in plasma and urine at the end the treatment were not affected by any treatment (results not shown). Antilithiasic activity A concentration-response curve was constructed using human urine, based on the effect of different doses of EEPam on the solution turbidity. Concentrations in both Piper extracts ranged from 0.00 to 5.00 mg/ml of urine (Fig. 5).
Fig. 5. Induction of CaOx crystallization measured by optical density (OD630 ) and number of crystals analyzed by Neubauer counting chamber, in healthy human urine by adding sodium oxalate in the absence and presence of increasing amounts of EEPam (A and B) extract with different concentrations (0.0625, 0.125, 0.250, 0.5, 1, 2.5 and 5 mg/l).
EEPam concentrations had increased crystal density and produced smaller crystals (Fig. 5 A-B). The increased absorbance indicates higher density of crystals. After crystallization assay in whole urine, the number of crystals was estimated by Neubauer counting chamber. In the presence of 0.25, 0.5 and 1 mg/ml of EEPam, there was an increase in the number of crystals compared with urine without EEPam (p < 0.01) (Fig. 5B). The alterations observed in the number of crystals were consistent in relation to increased turbidity index values, which confirm the data. Discussion
Fig. 4. Effect of the single oral administration with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. Urine samples were collected at 1, 2, 4, 6 and 24 h after treatment potassium levels were analyzed. Each bar represents the mean of five animals, and vertical lines show the S.E.M. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) (*p < 0.05; **p < 0.01; ***p < 0.001).
The traditional use of medicinal plants is common in many countries as an alternative primary health care (Bouanani et al., 2010). Several plants, including Piper species, specially P. amalago are used in the Brazilian folk medicine as diuretic drugs and for kidney diseases (Achenbach et al., 1986; Coimbra, 1994). This investigation showed that EEPam (125, 250 and 500 mg/kg) significantly increase urinary excretion of electrolytes Na+ and K+ and water in treated groups when compared to controls. Gasparotto Junior et al. (2012) showed that Tropaeolum majus has diuretic effect, which was associated to flavonoid isoquercitrin (Gasparotto Junior et al., 2012). The diuretic effect of Piper species investigated can be related to the presence of naturally bioactive compounds
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such as flavonols and other phenolic compounds (Achenbach et al., 1986; Domínguez et al., 1986). However, this result and the mechanism of action should be elucidated in further studies. Loop diuretics take action mainly by blocking luminal NaK-2Cl transporter in the thick ascending limb of the loop of Henle (Martinez-Maldonado and Cordova, 1990; Rose, 1991). Loop diuretics, as furosemide, induce several effects such as metabolic alkalosis, ototoxicity, hyperuricemia, hypomagnesaemia, allergic reactions, each others (Jackson et al., 1996). In this sense, the search for new compounds with fewer or no adverse effect has been achieved, particularly in medicinal plants, due to its fewer adverse effects (Wright et al., 2007). Thus, our findings suggest that ethanolic extract of P. amalago have some action on Loop of Henle. The administration of doses of EEPam induced an increase on urinary excretion within the interval of 24 h. On the other hand, the oral administration of furosemide induced a significant increase in diuresis in all intervals. Furosemide has fast action due to its bioavailability (Vargo et al., 1995). The time difference for the onset of the diuretic action of these substances is possibly related to the characteristics of the gastrointestinal absorption of the extract components. In addition, other pharmacokinetic considerations may be related to the duration of the diuretic effect as possible active metabolites in the extract or active components that have a long half-life, or even the prolonged action at the site of action (Holford and Sheiner, 2011). EEPam at doses of 125, 250 and 500 mg/kg increased the urinary excretion of Na+ and water. The effect of the extracts was similarly to those of furosemide, thus, their mechanisms might be the same. However, further studies should be carried out in order to support this hypothesis. Regarding the antilithiasic activity, the adsorption into renal tubular or pelvic wall, where crystals can accumulate, is probably essential for stone formation. If the sedimentation rate increases, the size of crystals consequently increases, then, all the steps of reducing formation and crystal growth are important in preventing the aggregation of crystals (Bouanani et al., 2010). A decrease in the size of crystals was observed in the in vitro crystallization of EEPam. Thus, it is suggested that this extract probably has preventive properties to urolithiasis. Similar results were observed by (Barros et al., 2003), in which Phyllanthus niruri extract also decreased the size of crystals, indicating that the use of this plant can interfere in urolithiasis. An in vivo study on urolithiasis showed that triterpenes protect against toxic manifestations induced by oxalate and also against the production of free radicals. The mechanism involved is the inhibition of the aggregation of calcium oxalate crystals (Malini et al., 2000). Based on the data obtained by Barros et al. (2006) suggested that the antilithiasic effect of Phyllantus niruri may be due to the presence of triterpenes (Barros et al., 2006). Brancalion et al. (2012) showed that the presence of abundant heteroside flavonoids and phenolic compounds in the Copaifera langsdorffii extract (Brancalion et al., 2012). This class of compounds may contribute to the antilithiasic effect. The in vitro crystallization results showed an effect against the formation of crystals. P. amalago specie has terpenes and flavonoids in its composition (Achenbach et al., 1986; Okunade et al., 1997), thus, it is suggested that this effect can be due to the presence of these compounds. However, the mechanism involved in the formation of crystals is still unknown. Further scientific data are necessary to confirm such effects. The crystal growth inhibition potentially facilitates its removal, which could contribute to the antilithogenic effect of the plant (Barros et al., 2003). This study showed an increased turbidity by turbidimetry, which is interpreted as an increase in the number of crystals, and, by microscopy, it was possible to confirm the reduction in the size of crystals. This result supports the hypothesis that EEPam species have an antilithiasic effect. The results indicate that
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this specie had a significant effect in changing the in vitro crystallization process in CaOx in order to inhibit crystal growth. However, further study should be carried out to confirm this effect. In summary, this study showed that oral administration of ethanolic extract of P. amalago increased the urinary excretion of electrolytes Na+ and K+ as well as the urinary volume. In relation to the antilithiasic activity, EEPam reduced the size of calcium oxalate crystals and increased the number of crystals in in vitro crystallization, suggesting a preventive effect. The present study supports the ethnopharmacological use of P. amalago as diuretic agents, although further studies are necessary to evaluate the mechanisms involved in the biological activity and safety following repeated exposure. Additionally, the therapeutic effect against renal stones is effective with P. amalago, once this specie showed an effect in both experiments. As the extract is not purified fractions, the results of experiments carried out have satisfactory potential for the development of promising drugs. Finally, these results stimulate further investigations to extract and identify the active chemical compounds responsible for the effects observed here. Acknowledgements The authors are grateful to Laboratory of Clinical Analysis of HU/UFGD. Prof. Elsie Franklin Guimarães (Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, RJ, Brazil). FUNDECT – Fundac¸ão de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul. FNDCT – Fundo Nacional de Desenvolvimento Científico e tecnológico, MCT – Ministério da Ciência e Tecnologia, CNPq – National Council for Technological and Scientific Development”, UEMS – Universidade Estadual de Mato Grosso do Sul and UFGD – Universidade Federal da Grande Dourados for financial support and fellowships. References Achenbach, H., Fietz, W., Worth, J., Waibel, R., Portecop, J., 1986. Constituents of tropical medicinal plants, IXX1 GC/MS-investigations of the constituents of Piper amalago – 30 new amides of the niperine-type. Planta Medica 52, 12–18. Alécio, A.C., Bolzani, V., Young, M.C.M.d.S., Kato, M.J., Furlan, M., 1998. Antifungal amide from leaves of Piper hispidum. Journal of Natural Products 61, 637–639. Allie-Hamdulay, S., Rodgers, A.L., 2005. Prophylactic and therapeutic properties of a sodium citrate preparation in the management of calcium oxalate urolithiasis: randomized, placebo-controlled trial. Urological Research 33, 116–124. Alves, E.O., Mota, J.H., Soares, T.S., Vieira, M.C., Silva, C.B., 2008. Levantamento etnobotânico e caracterizac¸ão de plantas medicinais em fragmentos florestais de Dourados-MS. Ciência e Agrotecnologia 32, 651–658. Atmani, F., Khan, S.R., 2000. Effects of an extract from Herniaria hirsuta on calcium oxalate crystallization in vitro. BJU International 85, 621–625. Atmani, F., Lacour, B., Daudon, M., 1996. Uronic-acid-rich protein: a new glycoprotein inhibiting the crystallization of calcium oxalate in vitro. Nephrologie 17, 157–162. Balaji, K.C., Menon, M., 1997. Mechanism of stone formation. The Urologic Clinics of North America 24, 1–11. Barros, M.E., Lima, R., Mercuri, L.P., Matos, J.R., Schor, N., Boim, M.A., 2006. Effect of extract of Phyllanthus niruri on crystal deposition in experimental urolithiasis. Urological Research 34, 351–357. Benjumea, D., Abdala, S., Hernandez-Luis, F., Pérez-Paz, P., Martin-Herrera, D., 2005. Diuretic activity of Artemisia thuscula, an endemic canary species. Journal of Ethnopharmacology 100, 205–209. Bouanani, S., Henchiri, C., Migianu-Griffoni, E., Aouf, N., Lecouvey, M., 2010. Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. Journal of Ethnopharmacology 129, 38–45. Brancalion, A.P., Oliveira, R.B., Sousa, J.P., Groppo, M., Berretta, A.A., Barros, M.E., Boim, M.A., Bastos, J.K., 2012. Effect of hydroalcoholic extract from Copaifera langsdorffii leaves on urolithiasis induced in rats. Urological Research 40, 1–7. Calle-Alvarez, J., 1983. Contribuición al estudio de algunas especies de la família Piperaceae. Revista Colombiana de Química e Farmácia 4, 47–57. Carrara, V., Sera, L.Z.d.S., Cardozo-Filho, L., Cunha-Júnior, E.F., Cortez, E.C., DAG, T.-S., 2012. HPLC analysis of supercritical carbon dioxide and compressed propane extracts from Piper amalago L. with antileishmanial activity. Molecules 17, 15–33. Carter, B.L., 2012. Guidelines for use of diuretics: a view from a member of JNC 7. Journal of Clinical Hypertension 14, 273–276.
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Diuretic and antilithiasic activities of ethanolic extract from Piper amalago (Piperaceae) Antônio da Silva Novaes a , Jonas da Silva Mota b , Andersson Barison c , Clebson Luiz Veber c , Fábio Juliano Negrão a , Candida Aparecida Leite Kassuya a , Márcio Eduardo de Barros a,d,∗ a
Department of Health Sciences, Federal University of Grande Dourados, MS, Brazil Department of Biodiversity Research, University of the State of Mato Grosso do Sul, Dourados, MS, Brazil c Department of Chemistry, Polytechnic Center, Federal University of Paraná Federal University, Curitiba, PR, Brazil d General Hospital, Federal University of Grande Dourados, MS, Brazil b
a r t i c l e
i n f o
Article history: Received 4 July 2013 Received in revised form 4 September 2013 Accepted 6 October 2013 Keywords: Piper amalago Diuretic activity Urolithiasic Piperaceae
a b s t r a c t Objective: Piper amalago is used in Brazilian folk medicine as diuretic and for the treatment of urinary calculus disease, although no scientific data have been described to support these effects. Thus, this study aims to evaluate the diuretic effects and antilithiatic activity of the ethanolic extract of P. amalago (EEPam). Materials and methods: Ethanolic extracts of P. amalago (125, 250 and 500 mg/kg) were orally administered in male Wistar rats (n = 5) and urinary excretion was measured at intervals of up to 24 h after administration. The antilithiasic effect of EEPam on calcium oxalate urolithiasis crystallization was examined in a turbidimetric model. Results: The oral administration of all doses of EEPam significantly increased urine output after 24 h when compared to control group. Moreover, the application of EEPam, induced an inhibitory effect on calcium oxalate crystallization. Conclusions: According to results, P. amalago extracts showed diuretic and natriuretic activity and antilithiasic effects. © 2013 Elsevier GmbH. All rights reserved.
Introduction Diuretic drugs are clinically used to the treatment of oedema, hypertension, urolithiasis and others diseases (Orjala et al., 1992; Pietrow and Karellas, 2006; Rose, 1991; Wright et al., 2007). Urolithiasis is a disturbance that affects kidney, which is considered a systemic disorder associated with bone loss and fractures, chronic kidney disease, hypertension, increased risk of coronary artery disease, metabolic syndrome, and type-2 diabetes mellitus (Sakhaee et al., 2012). In addition, diuretics drugs are also used in the treatment of these diseases (Carter, 2012). However, diuretic drugs produce several adverse effects such as diabetes, hypokalemia and others (Zillich et al., 2006) while antilithiasic drugs have only preventive and protective effect (Allie-Hamdulay and Rodgers, 2005; Erickson et al., 2011; Tseng and Preminger, 2011; Xu et al., 2011). In this context, the search for new agents with diuretic and antilithiasic properties is of interest for public health.
∗ Corresponding author at: Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Dourados/MS 79825-070, MS, Brazil. Tel.: +55 67 3410 2321; fax: +55 67 3410 2326. E-mail addresses: [email protected], [email protected] (M.E. de Barros). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.10.014
In Brazil, there are several plants that are frequently used in folk medicine as diuretic drugs such as Piper amalago. This plant is also used against cardiovascular problems like hypertension and renal disturbances like renal stones (Achenbach et al., 1986; Coimbra, 1994; Dias et al., 1984; Parmar et al., 1997). In addition, traditional use of P. amalago commonly known in Brazil as “jaborandi” is used to alleviate chest pain and inflammation (Achenbach et al., 1986; Domínguez and Alcorn, 1985; Domínguez et al., 1986; Parmar et al., 1997). Besides, the tea from the leaves of P. amalago is used in treatment of burns (Alves et al., 2008). A neurobehavioral study showed that the methanolic extract from leaves of P. amalago acts on the central nervous system, with anxiogenic properties in rats evaluated in the model of the elevated plus-maze model affecting locomotion and exploration in this model, but without inducing genetic toxicity (Lopes et al., 2012). In the state Mato Grosso do Sul, Brazil, Piper species are used for several disorders (Alves et al., 2008). An ethnobotanical survey in Dourados, MS, Brazil, found that of the families identified, 10.8% were from Piper species, showing a large amount of species from this genus in the region (Alves et al., 2008). Phytochemical investigations showed that plants of the family Piperaceae have several compounds such as esters, phenolic ethers, volatile oils, lignans, and pyrrolidine alkaloids
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(Gibbs, 1974). Regarding the genus Piper L. several phytochemical groups such as lignans, flavonoids, lactones, alkaloids (pyrrolidines and piperidines), butenolides and cyclohexane epoxide have been found (Calle-Alvarez, 1983; Nair and Burke, 1990; Sengupta and Ray, 1987). Considering that the drugs currently available for the treatment of renal disturbances show undesirable side effects and to the best of our knowledge, no scientific data about the activity of P. amalago in the renal system have been described. The aim of this study was to assess diuretic, natriuretic and kaliuretic activities of these medicinal plants. Furthermore, in vitro antilithiasic activity was also investigated. Materials and methods Plant material Leaves of P. amalago were collected in Dourados, MS, Brazil in August, 2008 and identified by Prof. Elsie Franklin Guimarães from Jardim Botânico Research Institute of Rio de Janeiro, RJ, Brazil. A voucher specimen (DDMS 4410), was deposited in the herbarium of the Federal University of Grande Dourados, MS, Brazil. Ethanolic extract and phytochemical analysis Dried at room temperature and powdered leaves of P. amalago (816 g) was extracted with ethanol 92% for 7 days by maceration. The solvent was then eliminated under reduced pressure to 1/6 of initial volume and then freeze-dried, yielding 4.2% (w/w). P. amalago ethanolic extract (30 g) was submitted to column chromatography over silica gel eluted with increasing concentrations of hexane in AcOEt. The chromatographic fractioning resulted in 1 (32.0 mg), 2 (11.5 mg), 3 (5.1 mg) and 4 (5 mg) (Fig. 1). The compounds were identified by exhaustive analyses of 1D and 2D NMR data and compared with literature data (Alécio et al., 1998; Carrara et al., 2012; Domínguez et al., 1985, 1986; Jacobs et al., 1999; Nascimento et al., 2012). During the experiments, the freeze-dried extract was dissolved in distilled water with Tween 80. Chemicals and general procedures Furosemide from Lasix, Sanofi-Aventis Farmacêutica, a highceiling loop diuretic, was used as reference drug (positive control). It was dissolved in distilled water prior to administration. EDTA, Tween 80, Sodium oxalate and Fluid Pack were of analytical grade. NMR data were acquired on a Bruker AVANCE III 400 NMR spectrometer operating at 9.4 Tesla, observing 1 H and 13 C nuclei at 400 and 100 MHz, respectively. Animals The experiments were performed with male adult 8-weekold Wistar rats provided by the vivarium of Federal University of Grande Dourados. The animals were maintained under a 12 h light–dark cycle, 60–80% humidity at 22 ± 1 ◦ C and received food and water ad libitum. Rats were handled in accordance with internationally accepted standard guidelines for use of animals. All procedures were approved by the Ethics Committee for research on humans and animals, under number 034/2011 and 011/2011, respectively. Diuretic activity assay Diuretic activity was determined according to Kau et al. (1984) method (Kau et al., 1984), with some modifications. Brief, rats after
Fig. 1. Chemical compounds found in leaves of Piper amalago (Piperaceae).
overnight fasting and water ad libitum were divided into five groups (n = 5). Before treatment, all animals received physiological saline (0.9% NaCl) at an oral dose of 5% body weight to impose a uniform water and salt load (Benjumea et al., 2005). Then, ethanolic extract forms P. amalago were orally administered to the animals at doses of 125, 250 and 500 mg/kg (b.w.). Negative control group received the same amount of distilled water while those from positive control received 10 mg/kg (b.w.) of furosemide. Immediately after administration, the animals were placed in metabolic cages. Urine was collected and measured at 1, 2, 4, 6, and 24 h. Cumulative urine excretion was calculated in relation to body weight and expressed as ml/100 g b.w. Electrolyte (Na+ , K+ ) concentrations were estimated from each urine sample of each rat at all time intervals using Roche 9180 Electrolyte Analyzer and expressed as mmol/100 g (b.w.) and pH was verified at the end of experiments, as well. The diuretic index (volume treated group/volume control group and saluretic (mmol/l treated group/mmol/l control group) was calculated.
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Table 1 Effect of single oral EEPam administration on urine volume and electrolyte excretion. Treatment
Vehicle Furosemide EEPam EEPam EEPam
Dose (mg/kg)
– 10 125 250 500
Urine volume (ml/100 g/24 h)
4.1 6.8 6.36 5.51 5.44
± ± ± ± ±
0.3 0.2*** 0.2*** 0.2* 0.4*
Diuretic indexa
– 1.67 1.54 1.34 1.32
Na+ (mmol/l/100 g/24 h)
477 682 660 590 667
± ± ± ± ±
10.1 23.8*** 14.1*** 37.9** 38.6***
K+ (mmol/l/100 g/24 h)
428 548 520 524 537
± ± ± ± ±
15.2 21.8*** 7.0*** 16.3*** 17.7***
Saluretic indexb Na+
K+
– 1.42 1.38 1.23 1.39
– 1.28 1.21 1.22 1.25
Data are expressed as mean ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001 in comparison with the control group (vehicle) using One-Way variance test (ANOVA) followed by Student–Newman–Keuls test. a Diuretic index = volume treated group/volume control group. b Saluretic index = mmol/l treated group/mmol/l control group.
Blood serum analysis After diuretic experiments, the animals were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) and blood samples (3–5 ml) collected in tubes containing EDTA (1 mg/ml) by puncture from renal vein. Plasma was obtained by 10 min centrifugation at 4000 rpm. The plasma was separated and stored at −20 ◦ C for further analysis. Na+ and K+ levels in plasma were determined by an automatic Roche, 9180 Eletrolyte Analyzer.
test, or Student’s t-test. P-values less than 0.05 were considered statistically significant. Graphs were drawn and statistical analysis was carried out using the GraphPad Prism software version 5.00 for Windows (GraphPad Software).
Antilithiasic activity
The phytochemical analysis of ethanolic extract from the leaves of P. amalago showed the presence of abundant pyrrolidide amides as well as chalcones and a flavonol (Fig. 1).
Calcium oxalate crystallization was induced in the urine obtained from healthy individuals. Human urine samples were obtained from six healthy subjects, three men and three women, with no personal or family history of kidney stone disease. Human urine samples were centrifuged at 5000 rpm for 8 min, the supernatant was then transferred to a clean tube, the pH was adjusted to 6.0 and it was filtered through a 0.22 m filter (Barros et al., 2003). The in vitro calcium oxalate crystallization protocol was modified, according with some methods described in literature (Atmani and Khan, 2000; Atmani et al., 1996; Balaji and Menon, 1997; Barros et al., 2003; Dussol and Berland, 1998; Robertson et al., 1968). Calcium oxalate precipitation was induced by adding 50 l of 0.1 M sodium oxalate per ml of urine (corresponding to 6.7 mg/ml), every 15 min (0, 15, 30 and 45 min) under shaking at 37 ◦ C, resulting in final urine concentration of 26.8 mg/ml. Each urine sample was divided into two aliquots, one of which was used as control (crystallization without Piper extract) while the other CaOx precipitation was induced in the presence of Piper extract, which was added to the sample 30 min before the crystallization process. Freezedried Piper amalago extract was resuspended in distilled water, filtered through 0.22 m filter, and used at final urine concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 2.5 and 5 mg/ml, based on a concentration-response curve. The crystals obtained were semiquantitative estimated by turbidity, immediately after crystallization. Aliquots of 100 l were loaded onto a 96 well microplate and the absorbance was measured on a plate reader (Thermoplate Reader), at 630 nm in quadruplicate and the average was used to calculate the turbidity index TI = (DOt × DOb)/DOb, where DOt is the mean sample absorbance after calcium oxalate precipitation and DOb is the mean sample absorbance before precipitation (Barros et al., 2003). The number and size of crystals were determined with aid of a Neubauer counting chamber. Statistical analysis The results are expressed as mean ± S.E.M. of experiments. Statistical significance was determined through one-way analysis of variance (ANOVA), followed by either Newman–Keuls test, Dunnet
Results Phytochemical analysis
Diuretic activity The effects of the oral administration of ethanolic extract of P. amalago on the urinary outputs are shown in Fig. 2 and Table 1. A significant increasing in urinary excretion (p < 0.001) was observed for 125 mg/kg of EEPam for 1, 2, and 24 h (p < 0.001), as well as for 500 and 250 mg/kg (p < 0.05) (Fig. 2). The effect of a single dose of reference diuretic, furosemide, induced a significant increase in the Na+ excretion at all times analyzed during the 24 h of experiment (P < 0.001). The amount of urinary electrolytes (Na+ and K+ ) was measured in all samples collected at 1, 2, 4, 6 and 24 h as shown in Figs. 3 and 4. The positive
Fig. 2. Time course of urinary volume excretion in rats treated with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. The urine volume was measured at 1, 2, 4, 6 and 24 h after treatment. Cumulative values are reported as mean ± SEM for five rats in each group. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) *p < 0.05; **p < 0.01; ***p < 0.001.
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Fig. 3. Effect of single oral administration with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. Urine samples were collected at 1, 2, 4, 6 and 24 h after treatment and sodium levels were analyzed. Each bar represents the mean of five animals, and vertical lines show the S.E.M. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) (*p < 0.05; **p < 0.01; ***p < 0.001).
control group revealed that the amount of Na+ and K+ significantly increased in all times evaluated. The EEPam dose of 125 mg/kg was the most potent, with a diuretic index of 1.54. Equally important, for doses of 250 and 500 mg/kg, the diuretic indexes were 1.34 and 1.32, respectively (Table 1). Na+ and K+ levels in plasma and urine at the end the treatment were not affected by any treatment (results not shown). Antilithiasic activity A concentration-response curve was constructed using human urine, based on the effect of different doses of EEPam on the solution turbidity. Concentrations in both Piper extracts ranged from 0.00 to 5.00 mg/ml of urine (Fig. 5).
Fig. 5. Induction of CaOx crystallization measured by optical density (OD630 ) and number of crystals analyzed by Neubauer counting chamber, in healthy human urine by adding sodium oxalate in the absence and presence of increasing amounts of EEPam (A and B) extract with different concentrations (0.0625, 0.125, 0.250, 0.5, 1, 2.5 and 5 mg/l).
EEPam concentrations had increased crystal density and produced smaller crystals (Fig. 5 A-B). The increased absorbance indicates higher density of crystals. After crystallization assay in whole urine, the number of crystals was estimated by Neubauer counting chamber. In the presence of 0.25, 0.5 and 1 mg/ml of EEPam, there was an increase in the number of crystals compared with urine without EEPam (p < 0.01) (Fig. 5B). The alterations observed in the number of crystals were consistent in relation to increased turbidity index values, which confirm the data. Discussion
Fig. 4. Effect of the single oral administration with different doses (0.125, 0.250, and 0.500 g/kg) of ethanolic extract of P. amalago (EEPam) and furosemide as reference drug. Urine samples were collected at 1, 2, 4, 6 and 24 h after treatment potassium levels were analyzed. Each bar represents the mean of five animals, and vertical lines show the S.E.M. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Newman–Keuls test) (*p < 0.05; **p < 0.01; ***p < 0.001).
The traditional use of medicinal plants is common in many countries as an alternative primary health care (Bouanani et al., 2010). Several plants, including Piper species, specially P. amalago are used in the Brazilian folk medicine as diuretic drugs and for kidney diseases (Achenbach et al., 1986; Coimbra, 1994). This investigation showed that EEPam (125, 250 and 500 mg/kg) significantly increase urinary excretion of electrolytes Na+ and K+ and water in treated groups when compared to controls. Gasparotto Junior et al. (2012) showed that Tropaeolum majus has diuretic effect, which was associated to flavonoid isoquercitrin (Gasparotto Junior et al., 2012). The diuretic effect of Piper species investigated can be related to the presence of naturally bioactive compounds
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such as flavonols and other phenolic compounds (Achenbach et al., 1986; Domínguez et al., 1986). However, this result and the mechanism of action should be elucidated in further studies. Loop diuretics take action mainly by blocking luminal NaK-2Cl transporter in the thick ascending limb of the loop of Henle (Martinez-Maldonado and Cordova, 1990; Rose, 1991). Loop diuretics, as furosemide, induce several effects such as metabolic alkalosis, ototoxicity, hyperuricemia, hypomagnesaemia, allergic reactions, each others (Jackson et al., 1996). In this sense, the search for new compounds with fewer or no adverse effect has been achieved, particularly in medicinal plants, due to its fewer adverse effects (Wright et al., 2007). Thus, our findings suggest that ethanolic extract of P. amalago have some action on Loop of Henle. The administration of doses of EEPam induced an increase on urinary excretion within the interval of 24 h. On the other hand, the oral administration of furosemide induced a significant increase in diuresis in all intervals. Furosemide has fast action due to its bioavailability (Vargo et al., 1995). The time difference for the onset of the diuretic action of these substances is possibly related to the characteristics of the gastrointestinal absorption of the extract components. In addition, other pharmacokinetic considerations may be related to the duration of the diuretic effect as possible active metabolites in the extract or active components that have a long half-life, or even the prolonged action at the site of action (Holford and Sheiner, 2011). EEPam at doses of 125, 250 and 500 mg/kg increased the urinary excretion of Na+ and water. The effect of the extracts was similarly to those of furosemide, thus, their mechanisms might be the same. However, further studies should be carried out in order to support this hypothesis. Regarding the antilithiasic activity, the adsorption into renal tubular or pelvic wall, where crystals can accumulate, is probably essential for stone formation. If the sedimentation rate increases, the size of crystals consequently increases, then, all the steps of reducing formation and crystal growth are important in preventing the aggregation of crystals (Bouanani et al., 2010). A decrease in the size of crystals was observed in the in vitro crystallization of EEPam. Thus, it is suggested that this extract probably has preventive properties to urolithiasis. Similar results were observed by (Barros et al., 2003), in which Phyllanthus niruri extract also decreased the size of crystals, indicating that the use of this plant can interfere in urolithiasis. An in vivo study on urolithiasis showed that triterpenes protect against toxic manifestations induced by oxalate and also against the production of free radicals. The mechanism involved is the inhibition of the aggregation of calcium oxalate crystals (Malini et al., 2000). Based on the data obtained by Barros et al. (2006) suggested that the antilithiasic effect of Phyllantus niruri may be due to the presence of triterpenes (Barros et al., 2006). Brancalion et al. (2012) showed that the presence of abundant heteroside flavonoids and phenolic compounds in the Copaifera langsdorffii extract (Brancalion et al., 2012). This class of compounds may contribute to the antilithiasic effect. The in vitro crystallization results showed an effect against the formation of crystals. P. amalago specie has terpenes and flavonoids in its composition (Achenbach et al., 1986; Okunade et al., 1997), thus, it is suggested that this effect can be due to the presence of these compounds. However, the mechanism involved in the formation of crystals is still unknown. Further scientific data are necessary to confirm such effects. The crystal growth inhibition potentially facilitates its removal, which could contribute to the antilithogenic effect of the plant (Barros et al., 2003). This study showed an increased turbidity by turbidimetry, which is interpreted as an increase in the number of crystals, and, by microscopy, it was possible to confirm the reduction in the size of crystals. This result supports the hypothesis that EEPam species have an antilithiasic effect. The results indicate that
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this specie had a significant effect in changing the in vitro crystallization process in CaOx in order to inhibit crystal growth. However, further study should be carried out to confirm this effect. In summary, this study showed that oral administration of ethanolic extract of P. amalago increased the urinary excretion of electrolytes Na+ and K+ as well as the urinary volume. In relation to the antilithiasic activity, EEPam reduced the size of calcium oxalate crystals and increased the number of crystals in in vitro crystallization, suggesting a preventive effect. The present study supports the ethnopharmacological use of P. amalago as diuretic agents, although further studies are necessary to evaluate the mechanisms involved in the biological activity and safety following repeated exposure. Additionally, the therapeutic effect against renal stones is effective with P. amalago, once this specie showed an effect in both experiments. As the extract is not purified fractions, the results of experiments carried out have satisfactory potential for the development of promising drugs. Finally, these results stimulate further investigations to extract and identify the active chemical compounds responsible for the effects observed here. Acknowledgements The authors are grateful to Laboratory of Clinical Analysis of HU/UFGD. Prof. Elsie Franklin Guimarães (Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, RJ, Brazil). FUNDECT – Fundac¸ão de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul. FNDCT – Fundo Nacional de Desenvolvimento Científico e tecnológico, MCT – Ministério da Ciência e Tecnologia, CNPq – National Council for Technological and Scientific Development”, UEMS – Universidade Estadual de Mato Grosso do Sul and UFGD – Universidade Federal da Grande Dourados for financial support and fellowships. References Achenbach, H., Fietz, W., Worth, J., Waibel, R., Portecop, J., 1986. Constituents of tropical medicinal plants, IXX1 GC/MS-investigations of the constituents of Piper amalago – 30 new amides of the niperine-type. Planta Medica 52, 12–18. Alécio, A.C., Bolzani, V., Young, M.C.M.d.S., Kato, M.J., Furlan, M., 1998. Antifungal amide from leaves of Piper hispidum. Journal of Natural Products 61, 637–639. Allie-Hamdulay, S., Rodgers, A.L., 2005. Prophylactic and therapeutic properties of a sodium citrate preparation in the management of calcium oxalate urolithiasis: randomized, placebo-controlled trial. Urological Research 33, 116–124. Alves, E.O., Mota, J.H., Soares, T.S., Vieira, M.C., Silva, C.B., 2008. Levantamento etnobotânico e caracterizac¸ão de plantas medicinais em fragmentos florestais de Dourados-MS. Ciência e Agrotecnologia 32, 651–658. Atmani, F., Khan, S.R., 2000. Effects of an extract from Herniaria hirsuta on calcium oxalate crystallization in vitro. BJU International 85, 621–625. Atmani, F., Lacour, B., Daudon, M., 1996. Uronic-acid-rich protein: a new glycoprotein inhibiting the crystallization of calcium oxalate in vitro. Nephrologie 17, 157–162. Balaji, K.C., Menon, M., 1997. Mechanism of stone formation. The Urologic Clinics of North America 24, 1–11. Barros, M.E., Lima, R., Mercuri, L.P., Matos, J.R., Schor, N., Boim, M.A., 2006. Effect of extract of Phyllanthus niruri on crystal deposition in experimental urolithiasis. Urological Research 34, 351–357. Benjumea, D., Abdala, S., Hernandez-Luis, F., Pérez-Paz, P., Martin-Herrera, D., 2005. Diuretic activity of Artemisia thuscula, an endemic canary species. Journal of Ethnopharmacology 100, 205–209. Bouanani, S., Henchiri, C., Migianu-Griffoni, E., Aouf, N., Lecouvey, M., 2010. Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. Journal of Ethnopharmacology 129, 38–45. Brancalion, A.P., Oliveira, R.B., Sousa, J.P., Groppo, M., Berretta, A.A., Barros, M.E., Boim, M.A., Bastos, J.K., 2012. Effect of hydroalcoholic extract from Copaifera langsdorffii leaves on urolithiasis induced in rats. Urological Research 40, 1–7. Calle-Alvarez, J., 1983. Contribuición al estudio de algunas especies de la família Piperaceae. Revista Colombiana de Química e Farmácia 4, 47–57. Carrara, V., Sera, L.Z.d.S., Cardozo-Filho, L., Cunha-Júnior, E.F., Cortez, E.C., DAG, T.-S., 2012. HPLC analysis of supercritical carbon dioxide and compressed propane extracts from Piper amalago L. with antileishmanial activity. Molecules 17, 15–33. Carter, B.L., 2012. Guidelines for use of diuretics: a view from a member of JNC 7. Journal of Clinical Hypertension 14, 273–276.
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