Ethnopharmacological investigation of the diuretic and hemodynamic properties of native species of the Brazilian biodiversity

Author’s Accepted Manuscript Ethnopharmacological investigation of the diuretic and hemodynamic properties of native species of the Brazilian biodiversity Thiago Buno Lima Prando, Lorena Neris Barboza, Francielly Mourão Gasparotto, Valdinei de Oliveira Araújo, Cleide Adriane SIgnor Tirloni, Lauro Mera de Souza, Emerson Luiz Botelho Lourenço, Arquimedes Gasparotto Junior

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To appear in: Journal of Ethnopharmacology Received date: 26 May 2015 Revised date: 22 July 2015 Accepted date: 23 August 2015 Cite this article as: Thiago Buno Lima Prando, Lorena Neris Barboza, Francielly Mourão Gasparotto, Valdinei de Oliveira Araújo, Cleide Adriane SIgnor Tirloni, Lauro Mera de Souza, Emerson Luiz Botelho Lourenço and Arquimedes Gasparotto Junior, Ethnopharmacological investigation of the diuretic and hemodynamic properties of native species of the Brazilian biodiversity, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2015.08.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ethnopharmacological investigation of the diuretic and hemodynamic properties of native species of the Brazilian biodiversity

Thiago Buno Lima Prandob; Lorena Neris Barbozab; Francielly Mourão Gasparottoa; Valdinei de Oliveira Araújob; Cleide Adriane SIgnor Tirlonia; Lauro Mera de Souzac*; Emerson Luiz Botelho Lourençob; and Arquimedes Gasparotto Juniora*

a

Laboratório de Farmacologia Cardiovascular, Faculdade de Ciências da Saúde,

Universidade Federal da Grande Dourados, Dourados, MS, Brasil b

Laboratório de Farmacologia e Toxicologia de Produtos Naturais, Universidade

Paranaense, PR, Brazil c

Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná,

Curitiba, PR, Brazil

*Corresponding author at: 1: Laboratório de Farmacologia Cardiovascular, Faculdade de Ciências da Saúde, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum, km 12, P.O. Box 533, 79.804-970 Dourados, MS, Brazil. Tel.: +55 (67) 3410-2333, Fax: +55 (67) 3410-2321. E-mail address: [email protected] (A. Gasparotto Junior).

2: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, P.O. Box 19046, 81.531-980, Curitiba, PR, Brazil. Tel.: +55 (41) 3361-1577, Fax: +55 (41) 3266-2042. E-mail address: [email protected] (L.M. de Souza).

Abstract Ethnopharmacological relevance: Although Echinodorus grandiflorus, Cuphea carthagenensis, and Phyllanthus tenellus infusions are used in Brazilian folk medicine due to their possible diuretic effect, none of these species was critically investigated as a diuretic drug. So, the aim of this study was to evaluate the possible acute diuretic activity of ethanol soluble fractions (ES) obtained from these species and assess the relationship between renal cortical blood flow and their antioxidant and hypotensive activity using normotensive Wistar rats. Material and methods: The preparation obtained from Echinodorus grandiflorus (ES-EG), Cuphea carthagenensis (ES-CC), and Phyllanthus tenellus (ES-PT) infusions was orally administered in a single +

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dose to rats. Urine excretion rate, pH, density, conductivity and Na , K , Cl andHCO3 contents were measured in the urine of saline-loaded animals. Concentration of electrolytes, total protein, urea, creatinine, and angiotensin converting enzyme (ACE) activity were evaluated in collected serum. The involvement of the renal cortical blood flow and antioxidative activity in the hypotensive and diuretic effects was also determined. +

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Results: Water and Na , Cl and Na excretion rates were significantly increased by ES-EG, while urinary bicarbonate excretion was reduced. Moreover, ES obtained from Echinodorus grandiflorus was able to significantly increase renal blood flow and reduce mean arterial pressure and oxidative stress in “in vitro” and “in vivo” models. All other parameters evaluated were not affected by any treatment. Conclusion: The results presented here shown that the ES-EG obtained from Echinodorus grandiflorus leaves shown a significant diuretic and hypotensive activity and suggest that these effects could be related with an important renal and systemic vasodilator effect. In addition, it was shown for the first time that the pharmacological effects of ES obtained from Phyllanthus tenellus and Cuphea carthagenensis do not support its popular use as a diuretic agent.

Keywords: Echinodorus grandiflorus; Cuphea carthagenensis; Phyllanthus tenellus; diuretic; vasodilator; hypotensive

1. Introduction The World Health Organization (WHO) estimates that high blood pressure (hypertension) is responsible for at least 45% of deaths due to heart diseases, and 51% of deaths due to stroke worldwide (WHO, 2013). Moreover, approximately 40% of adults aged 25 and above had been diagnosed with hypertension; the number of people with the condition rose from 600 million in 1980 to 1 billion in 2008, classifying hypertension as one of the most important global public health problems (WHO, 2011).

Hypertension prevention and control is complex and requires the engagement of patients, families, providers, healthcare systems and communities. This includes expanding patient and healthcare provider awareness, changing lifestyles (healthy diet, avoiding harmful use of alcohol, physical activity, stopping tobacco use, and stress management), high level of medication adherence, and adequate follow-up (Chobanian et al., 2003; Lloyd-Jones et al., 2010). Studies have shown that a substantial proportion of hypertensive patients do not have controlled blood pressure levels, and the major reason is the poor adherence to antihypertensive medications (Borzecki et al., 2005). Older age, living alone, and perception related to treatment control were significant independent factors associated with better medication adherence. Moreover, cultivating positive beliefs that hypertension is controlled by treatment is one of the most appropriate ways for adequate control of this pathology (Marshall et al., 2012). In this context, the socio-cultural appeal from medicinal plants, transferred by generations, translates an idea of reliability and safety of these herbal remedies, contributing to improve the therapeutic arsenal and helping adherence to antihypertensive medications. Thus, the popular culture is used in the identification of medicinal native species that can contribute to conventional treatment and encourage the belief that hypertension control is possible and might provide additional benefit. In South America, several natural products are used as antihypertensive agents primarily due to their diuretic activity. Nevertheless, very few native species have been critically investigated despite the immense biodiversity available in these countries. As an example, throughout South America, and especially in Brazil, Echinodorus grandiflorus (Cham. & Schltr.) Micheli, Cuphea carthagenensis (Jacq.) J.F. Macbr., and Phyllanthus tenellus Roxb. are widely used as diuretic, hypotensive and

antihypertensive drugs. Nevertheless, these species require a thorough ethnopharmacological investigation due to their extensive popular use as diuretics (Vendruscolo and Mentz, 2006; Lorenzi and Matos, 2008; Bolson et al., 2015). Echinodorus grandiflorus (Alismataceae) is an endemic species of Brazil. It is popularly known as “chapéu de couro” and it sporadically grows on various humid soils as support vegetative cover. The parts used as medicine are leaves that are habitually used as a hot water infusion orally administered. Traditionally, this plant has been used as diuretic, hypotensive, in the prevention of arteriosclerosis, depurative, antiinflammatory and analgesic (Lorenzi and Matos, 2008; Brugiolo et al., 2009; Bolson et al., 2015). This species have been shown to contain several fatty acids, diterpenoids, phenolic acids, flavonols, alkaloids, saponins and tannins (Brugiolo et al., 2009; Garcia et al., 2010). Several pharmacological studies performed with different extracts from the leaves of Echinodorus grandiflorus have shown anti-inflammatory and analgesic (Dutra et al., 2006), antihypertensive (Lessa et al., 2008), vasodilator (Tibiriçá et al., 2007), antimicrobial (Souza et al., 2004) and immunosuppressive effects (Pinto et al., 2007). Cuphea carthagenensis (Jacq.) J.F. Macbr. is a medicinal plant belonging to family Lythraceae, and popularly known in Brazil as “sete sangrias”. This species is found throughout South America, especially in the southern states of Brazil. The leaves of this plant are recommended in folk medicine to promoting and treating various diseases including fever, constipation, edemas, hypertension, atherosclerosis and circulatory disorders (Vendruscolo and Mentz, 2006; Lorenzi and Matos, 2008). Some constituents of this plant were detected to be triterpenes such as carthagenol (Gonzalez et al., 1994),tannins, proanthocyanidins, and flavonoids, including quercetin and their glycosylated derivatives (Krepsky et al., 2010; 2012).Recent pharmacological studies have shown that the crude extract obtained from this species was able of inhibit the in

vitro activity of the angiotensin converting enzyme (ACE) (Braga et al., 2000), and induce arterial vasodilation in rat aorta preparations (Schuldt et al., 2000; Krepsky et al., 2012). Moreover, the hypocholesterolemic effect (Biavatti et al, 2004) and radical scavenger activities of the crude extract and fractions obtained from Cuphea carthagenensis have also been reported (Schuldt et al., 2004). Phyllanthus tenellus Roxb. (Phyllantaceae) is an herbaceous plant that occurs practically throughout the Brazilian territory and is popularly known as "quebra pedras". This species is widely used in Brazilian traditional or folk medicine as a diuretic and are also indicated in the treatment of jaundice, hepatitis, kidney stones and diabetes (Calixto et al., 1998; Bolson et al., 2015; Tribess et al., 2015). Previous phytochemical studies conducted with Phyllanthus tenellus leaves have demonstrated the presence of various polyphenolic compounds such as condensed tannins, flavonoids and lignans (Calixto et al., 1998; Huang et al., 2003; Sprenger and Cass, 2013). Preclinical studies have shown positive effects in the treatment of hepatitis and urolithiasis (Calixto et al., 1997). In addition, antiviral (Shead et al., 1992), antibacterial (Oliveira et al., 2007), and analgesic effects (Santos et al., 1994) have also been reported. The aim of this work was to determine the pharmacological rationale for the empirical use of Echinodorus grandiflorus, Cuphea carthagenensis, and Phyllanthus tenellus as diuretic agents. The present study represents the first study to investigate the diuretic properties of a water infusion obtained from these species and relate this pharmacological activity with the effects on renal cortical blood flow in Wistar rats.

2. Materials and methods 2.1. Drugs Hydrochlorothiazide (HCTZ) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride was obtained from Merck (Darmstadt, Germany). All other reagents were obtained in analytical grade.

2.2. Phytochemical study 2.2.1. Plant material The aerial parts of Cuphea carthagenensis and Phyllanthus tenellus and leaves of Echinodorus grandiflorus were collected in February, 2014 from the botanical garden of the Universidade Paranaense (UNIPAR) (Umuarama, Brazil) at 430 m altitude above sea level (S23º47’55–W53º18’48). A voucher Cuphea carthagenensis and Phyllanthus tenellus specimens are cataloged in the Herbarium of the Universidade Paranaense (HEUP) under number 2403 and 2401, respectively. Echinodorus grandiflorus was cataloged in the Herbarium of the Universidade Estadual de Maringá (HUEM) under number 20810.

2.2.2. Preparation of aqueous extracts The plant material was air-dried in an oven at 37 °C for 5 days, and then cut and pulverized. The extracts were obtained by infusion in a similar manner to that used popularly in Brazil (Bolson et al., 2015). 1 L of boiling water was poured over 60 g of dried ground leaves; the container was closed, and the extraction was allowed to proceed until room temperature was reached (~ 6 h). The infusion extract was treated with 3 volumes of EtOH, which gave rise to a precipitate and an ethanol soluble fraction (ES). All preparations were freeze-dried and maintained at room temperature until

analyses. The aerial parts of Cuphea carthagenensis and Phyllanthus tenellus yielded 16% and 15%, respectively, whereas Echinodorus grandiflorus leaves yielded 13%.

2.2.3. Sample analysis (liquid chromatography-mass spectrometry) The samples were examined by ultra-high performance liquid chromatography (UPLCTM-Acquity, Waters Co., São Paulo, Brazil) composed by a binary pump and a photodiode array (PDA) detector. Samples at 2 mg/mL concentration were prepared in H2O-MeOH (7:3 v/v) and the analysis was carried out on a reversed-phase HSS-C18 column (2.1 x 100 mm with 1.7 µm of particle size, Waters Co.). The binary solvent was composed of (A) 0.1% aqueous formic acid (v/v) and (B) methanol. The linear solvent gradient at a flow rate of 0.4 mL min-1 was developed by increasing solvent percentage (B) from 0% to 38% at 10 min, then to 60% at 13 min and returning to initial condition at 14 min, re-equilibrated for 3 min (Carlotto et al., 2015). The column was heated at 60 oC and samples kept at room temperature (22 oC). The injection volume was 2 µL and compounds were detected at  200-400 by high resolution mass spectrometry (HR-MS). HR-MS analyses were developed on an electrospray ionization mass spectrometry (ESI-MS) LTQ-Orbitrap-XL (Thermo-Scientific, Waltham, USA), operating in the negative ionization at atmospheric pressure ionization (API). The source temperature was 350 oC and N2 stream was used for sample desolvation with sheath gas flow rate at 60 arbitrary units (abu) and auxiliary at 20 abu. The ionization parameters were: spray voltage of 3.5 kV, capillary of -20 V and tube lens of -130 V. For mass accuracy, external calibration was performed (100 to 2000 m/z) and resolution was set at 30,000 FWHM (at m/z 400) in LC-MS mode. Acquisition was obtained in total ion current (TIC) mode, with mass range of m/z 100–1000. Compound fragmentation was obtained by collision-induced dissociation (CID) using helium and energy of 20eV.

2.3. Pharmacological tests 2.3.1. Animals Male Wistar rats (250–300 g) were used in experiments. The animals were provided by Federal University of Paraná (UFPR, Curitiba/PR, Brazil), and kept at temperature- and light-controlled room (22 ± 2 °C; 12-h light/dark cycle) with free access to water and food. All experimental procedures adopted in this study were previously approved by the Institutional Ethics Committee of Universidade Paranaense (UNIPAR, Brazil; protocol number 25454/2014).

2.3.2. Acute diuretic activity The diuretic activity assessment was performed according to methods previously described (Kau et al., 1984) with some modifications (Gasparotto Junior et al., 2009). Rats were fasted overnight (12 h) with free access to water. Before treatments, all animals received an oral load of isotonic saline (0.9% NaCl, 5 mL/100 g) to impose a controlled water and salt balance. Subsequently, different groups of rats were orally treated with ES-EG (30, 100 or 300 mg/kg),ES-PT (30, 100 or 300 mg/kg), ES-CC (30, 100 or 300 mg/kg), or HCTZ (10 mg/kg). Control rats received the same volume of deionized water, the vehicle used to dissolve the extracts. Immediately after treatment, rats were placed in metabolic cages. Urine was collected in a graduated cylinder and its volume was recorded for 8 h (expressed as mL/100 g). At the end of the experimental period, animals were sacrificed and their blood was collected for biochemical analysis.

2.3.2.1. Analytical procedures For serum analysis, blood samples were collected in conical tubes after

decapitation. Plasma and serum were obtained by centrifugation (2000 rpm, 10 minutes, 4 ºC), and stored at -20 ºC until analysis. Urinary and plasmatic Na+ and K+ levels were quantified by Q398112 flame photometry (Quimis Instruments, Diadema, Brazil). Cl- and HCO3- concentrations were quantified by titration. pH and conductivity were directly determined on fresh urine samples using Q400MT pH-meter and Q795M2 conductivity meter (Quimis Instruments, Diadema, Brazil), respectively. Density was estimated by weighing with a Mettler AE163 (± 0.1 mg) analytical scale of urine volume measured with a Nichiryo micropipette. Plasma total protein, urea and creatinine concentrations were determined by enzymatic method using BM/Hitachi 912 automated analyzer (Cobas Mira, Roche, Indianapolis, USA). Serum Angiotensin Converting Enzyme (ACE) activity was measured by methods previously described (Santos et al., 1985). Additionally, excretion load (El) of sodium, potassium, chloride and bicarbonate was obtained according to the equation below (1): (1) El=Ux x V

Ux: Electrolytes concentration (mEq/L) V: Urinary flow (mL/min) Results were expressed as μEq/min/100g

2.3.3. Hemodynamic study 2.3.3.1. Direct blood pressure and heart rate measurement Normotensive male Wistar rats were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), intramuscularly administered and supplemented at 45 to 60 min intervals. A polyethylene catheter was inserted into the right femoral vein for drug

administration. Immediately after venous cannulation, a bolus injection of heparin (30 IU) was i.v administered. Animals were allowed to breath spontaneously through a tracheotomy. The left carotid artery was cannulated and connected to a pressure transducer coupled to a PowerLab® recording system, and an application program (Chart, v 4 .1; all from ADI Instruments; Castle Hill, Australia) recorded both mean arterial pressure (MAP) and heart rate (HR) (Gasparotto Junior et al., 2011). After this procedure, different groups of rats received ES-EG (30, 100 or 300 mg/kg), ES-PT (30, 100 or 300 mg/kg), or the ES-CC (30, 100 or 300 mg/kg) intraduodenally. The control group received vehicle intraduodenally at a constant volume of 100 µL/100g of body weight. The changes in MAP and HR were recorded for 45 min after treatments. Each animal received only one of the substances studied. At the end of experiments, animals were sacrificed with an overdose of thiopental (over 40 mg/kg, i.v.).

2.3.3.2. Renal cortical blood flow (RCBF) Renal cortical blood flows were measured according to previous methods (Fleming et al., 2000). After preparation for PAM measurement (as described above), a standard plastic tube (DP2b, Moor Instruments, Axminster, UK) was placed on the ventral surface of the kidney (renal capsule below 2 mm) coupled to a PowerLab® recording system, and an application program (Chart, v 4 .1; all from ADI Instruments; Castle Hill, Australia). This system measures tissue perfusion by the number of cells moving and their mean velocity within an area of 1 mm3 beneath the tip of each probe. The laser-Doppler signal (LDS) is independent of the cell movement direction. After this procedure, different groups of rats received ES-EG (30, 100 or 300 mg/kg), ES-PT (30, 100 or 300 mg/kg), ES-CC (30, 100 or 300 mg/kg) or vehicle intraduodenally, at constant volume of 100 µL/100g of body weight. The changes in RCBF were recorded

for 45 min after treatments. Each animal received only one of the substances studied. Proper location of the optical fibers within the kidney was verified at the end of the experiment by inspecting the location of the fiber tips.

2.3.4. Oxidative stress scavenger assays 2.3.4.1. DPPH radical scavenging activity The method of capturing 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was used to determine the antioxidant activity as described by Gupta and Gupta (2011) with some modifications. Various ES-EG, ES-PT or ES-CC (1000, 500, 100, 50, 25, 10, 5, 1 and 0.1 mg/mL) concentrations were homogenized in 1.8 ml of a methanol DPPH solution (0.11 mM). Reading was performed using spectrophotometer at wavelength of 517 nm after 30 minutes incubation with and without light room temperature. Antioxidant ascorbic acid was used as a standard at the same concentrations as the extract. Methanol was used as blank. IC 50 values were calculated from the regression equation prepared for the different concentrations of both methanol and aqueous extracts.

2.3.4.2. Hemolytic activity and protection against 2,29 –Azobis (2-amidinopropane) dihydrochloride (AAPH)-induced hemolysis Protection against lipid peroxidation was assessed by the hemolysis protection method induced by AAPH described by Valente et al. (2011). A solution of 10% erythrocytes was prepared in saline (He) and pre-incubated at 37 °C for 30 min in the presence of different ascorbic acid, ES-EG, ES-PT or ES-CC (1000, 300, 100, 30, 10 μg/mL) concentrations. Then, 50 mM AAPH solution was added. Total hemolysis was induced by incubating He with distilled water. Basal hemolysis caused by aqueous extract (ES) was assessed by incubating He with the extract without the presence of a

hemolysis inducer, and the negative control was assessed in He incubated only with 0.9% NaCl. After adding AAPH, aliquots were collected at 60, 120, 180 and 240 min and read at 540 nm. Three independent experiments were performed in triplicate.

2.3.4.3. Nitric oxide radical assay Quantitative measurements of the nitric oxide radical scavenging property were carried out using the Griess Illosvoy reaction (Kumar et al., 2008). ES-EG, ES-PT and ES-CC at various concentrations (1000, 300, 100, 30, 10 μg/mL) were mixed with 2 mL sodium nitroprusside (10 mM) in standard phosphate saline buffer (50 mM, pH 7.4) and incubated at room temperature for 3 hours. After the incubation period, samples were diluted with of 0.5 mL of Griess reagent. The absorbance of the color developed during nitrite diazotization with sulphanilamide and its subsequent coupling with N-(1-Naphthyl) ethylenediamine dihydrochloride was measured at 550 nm on spectrophotometer. Ascorbic acid was used as standard. IC50 values were calculated from the regression equation prepared for the different aqueous extract concentrations.

2.3.4.4. Serum nitrate/nitrite determination (NOx) Plasma nitrite concentration was determined by enzymatically reducing nitrate using nitrate reductase enzyme (Schmidt et al., 1989). After evaluation of the acute diuretic activity (8h after treatments), plasma samples were collected from rats, deproteinized with zinc sulfate (30 mmol) and diluted at 1:1 with Milli-Q water. For the conversion of nitrate into nitrite, samples were incubated at 37 °C for 2 hours in the presence of nitrate reductase expressed in E. coli. After the incubation period, samples were centrifuged (800 g, 10 minutes) to remove bacteria. Then, 100 µL of supernatant were mixed with an equal volume of Griess reagent (1% sulfanilamide in 10%

phosphoric acid/0.1% alpha-naphthyl-ethylenediamine in Milli-Q water) in a 96-well plate and read at 540 nm in a plate reader. Standard nitrite and nitrate curves (0-150 mM) were simultaneously performed.

2.3.5. Statistical analysis Results are expressed as mean ± standard error of the mean of five animals in each group. Statistical evaluation was carried out by analysis of variance (ANOVA) followed by Dunnett's test. P-value less than 0.05 was considered statistically significant. Graphs were drawn and statistical analysis was carried out using the GraphPad Prism software version 5.0 for Mac OS X (GraphPad Software, San Diego, CA, USA).

3. Results 3.1. Phytochemical characterization ES-EG obtained from Echinodorus grandiflorus was analyzed by LC-MS, resulting in a great variety of phenolic compounds, mainly flavonoids C-glycosides (Figure 1A), which were confirmed by collision induced dissociation (CID-MS) analysis, being in accordance with previous findings (Schnitzler, et al 2007; Garcia, et al 2010). The presence of this compounds was confirmed by HR-MS, which gave negative ions consistent with mono-C-flavonoids, such as isoorientin at m/z 447.09322 (Rt 8.73), swertiajaponin at m/z 461.10884 (Rt 8.96), isovitexin at m/z 431.09826 (Rt 9.72), swertisin at m/z 445.09322 (Rt 10.01) and isoorientin-dimethylether at m/z 475.12439 (Rt 10.36). Flavonoids di-C-glycosides, such as isoorientin-rhamonoside at m/z 593.15120 (Rt 8.91), isoorientin-rhamnoside-dimethylether at m/z 621.18254 (Rt 10.53), isovitexin-pentoside at m/z 563.14049 (Rt 8.60), swertiajaponin-rhamonoside at m/z

607.16684 (Rt 10.26), isovitexin-rhamonoside at m/z 577.15633 (Rt 9.86), and swertisin-rhamnoside at m/z 591.17193 (Rt 10.22) were also present in this extract. At lower abundance, feruloyl-tartaric acid (m/z 325.05639, Rt 5.71) and diferuloyl-tartaric acid (m/z 501.10380, Rt 10.91 and 11.49) also appeared in the extract. LC-MS analysis of ES-PT obtained from Phyllanthus tenellus showed the presence of several hydrolysable tannins and some flavonoids glycosides (Figure 1B). HR-MS analysis revealed the presence of several ellagitannins at m/z 913.19088 (Rt 4.77), m/z 969.08769 (Rt 7.67), and m/z 923.08140(Rt 10.99), and hexahydroxydiphenoylglucopyranoside isomers at m/z 633.06710 (Rt 2.28), m/z 633.06560 (Rt 2.79) and m/z 633.07358 (Rt 6.44), which had similar fragments on CID-MS analysis. Flavonoids glycosides such as myricetin-rhamnoside (myricetrin) at m/z 463.08803 (Rt 9.30), quercetin-rutinoside m/z 609.14631 (Rt 9.80), and kaempferol-rutinoside m/z 593.15153 (Rt 10.88) were also observed. This composition is in accordance with Sprenger and Cass (2013). On the other hand, ES-CC analysis revealed the abundant presence of free flavonoids such as quercetin m/z 301.03555 (Rt 10.04) and glycosides, such as quercetin-hexoside m/z 463.08808 (Rt 9.21), quercetin-hexosyl-rhamnoside m/z 609.14593 (Rt 9.87), quercetin-pentoside m/z 433.07756 (Rt 10.55) and kaempferolrutinoside m/z593.15112 (Rt 10.99) (Figure 1C). Moreover, the presence of citric acid at m/z 191.01965 (Rt 0.96) was observed. The major peak at 9.73 min gave the ion at m/z 477.06754 with a main fragment on CID-MS at m/z 301.033, consistent with quercetin, but linked to an unknown group.

3.2. ES obtained from Echinodorus grandiflorus induces diuresis, natriuresis and increases renal cortical blood flow in normotensive Wistar rats Treatment with a single dose of ES-EG (100 and 300 mg/kg) significantly increased diuresis after 8 h (Table 1). The total volume of urine measured at 8 h in ES-EG-treated animals was 4.6 ± 0.2 and 5.4 ± 0.3 mL/100g, respectively; while the urinary volume observed in the control group at this same time was 3.2 ± 0.3 (p<0.05). The cumulative urinary volume found in ES-EG -treated animals (100 and 300 mg/kg) was not different from that obtained in animals treated with HCTZ. The effects of acute treatment of ES-EG (30, 100 and 300 mg/kg) on electrolyte excretion in urine collected at 8 h after treatments are presented in Table 2. Excreted sodium, potassium and chloride load was significantly increased in groups treated with ES-EG (100 and 300 mg/kg) when compared to controls treated with vehicle alone. Similarly, urine conductivity was significantly increased after treatment with the ES-EG at a dose of 300 mg/kg. Moreover, ES-EG presented an interesting sparing effect on bicarbonate in at all doses used, while group treated with hydrochlorothiazide showed high amounts of this electrolyte in the urine. All other urinary (pH and density; Table 1) or serum parameters (sodium, potassium, urea, creatinine, total protein, and ACE activity) showed no significant differences when compared to the control group. In addition to the above data, a significant increase in renal cortical blood flow was observed after acute administration of ES-EG (100 and 300 mg/kg). The data showed an increase ranging from 12.5 to 25% respectively, when compared to groups treated with vehicle alone (Figure 2A).

3.3. ES obtained from Cuphea carthagenensis and Phyllanthus tenellus was not able to induce any changes in renal function or cortical blood flow of Wistar rats Tables 1 and 2 showed the urine volume and electrolyte excretion produced in 8 hours after treatment in the different experimental groups. There was no statistically significant increase in urinary volume or electrolyte excretion in all animals receiving ES-CC or ES-PT throughout the experimental period. Moreover, acute ES-CC or ES-PT administration did not induce any significant changes in renal blood flow when compared to animals treated with vehicle alone (Figure 2B-C).

3.4. ES-EG induces significant hypotension after acute administration while ES-CC and ES-PT do not change blood pressure in normotensive rats The basal MAP recorded in anesthetized rats after the stabilization period and before the administration of any drug was 104.5 ± 2.3 mm Hg. The intraduodenal administration of ES-EG (300 mg/kg) caused a significant reduction (~15 mmHg; p<0.01) in MAP (Figure 2D). On the other hand, intraduodenal administration of ES-CC or ES-PT was not able to induce any hypotensive effects (Figure2E-F), with values similar to group treated with vehicle alone. In addition, none of the extracts changed heart rate (HR) (data not shown).

3.5. ES-EG induces antioxidative properties and nitric oxide production in Wistar rats In order to assess the contribution of the antioxidant properties of ES-EG, ES-CC and ES-PT in the renal and hemodynamic responses, initial screening with three "in vitro" tests was performed. Firstly, the ability of extracts to capture the DPPH free radical was evaluated. Secondly, the potential for AAPH-induced hemolysis inhibition was measured, and finally, their nitric oxide radical scavenging properties were also

evaluated. Data have shown that all extracts (ES-EG, ES-CC and ES-PT) were able to present DPPH radical scavenging activity with IC50 values estimated in 102 ± 4.3, 18 ± 4.1 and 6 ± 2.9 μg/mL, respectively (Table 3). After initial confirmation of the antioxidant activity, the effect of ES-EG, ES-CC and ES-PT on human erythrocytes was evaluated. All tested extracts did not induce hemolysis after incubation for 240 min (Figure 3), indicating that, at the concentrations evaluated, the extract is not toxic to normal cells. Moreover, during the 240-min incubation with hemolysis inducer AAPH, ES obtained from Echinodorus grandiflorus showed significant antioxidant activity protecting cells from cell lysis at concentrations of 300 and 1000 μg/mL, showing higher activity compared to extracts from Cuphea carthagenensis and Phyllanthus tenellus (Figure 3A-C). IC50 and maximum activity of nitric oxide radical scavenging of standard antioxidants and ES-EG, ES-CC and ES-PT are shown in Table 3. Although all tested extracts show antioxidant properties, only ES obtained from Echinodorus grandiflorus was able to induce maximal inhibitory effect at around 90%, with estimated IC50 value of 90 μg/mL. Considering the diuretic, antioxidant and hemodynamic properties of ES obtained from Echinodorus grandiflorus; we chose to measure the "in vivo" concentration of a nitric oxide bioavailability marker. So, the acute administration of ES-EG (100 and 300 mg/kg), but not ES-CC and ES-PT, significantly increased the serum nitrate levels when compared to the control group (ES-EG 100: 75.6 ± 4.7 μM; ES-EG 300: 88.8 ± 3.9 μM; control: 50.8 ± 6.6 μM; p<0.05) (Figure 3D-F), showing that the "in vitro" antioxidant effects observed with ES-EG appear also be evident in Wistar rats.

4. Discussion and conclusions There is strong evidence about the pharmacological benefits of Echinodorus grandiflorus, Cuphea carthagenensis, and Phyllanthus tenellus in the treatment of various cardiovascular disorders (Braga et al., 2000; Schuldt et al., 2000; Lessa et al., 2008). These species have been used in Brazilian folk medicine as an adjunct to conventional treatments to produce diuretic effects for decades (Lorenzi and Matos, 2008; Bolson et al., 2015). Nevertheless, to the present moment, there are no studies using a suitable experimental model, which validates their use as a diuretic as they are used in traditional medicine. Thus, the results obtained in this study bring to light evidence and show for first time that only infusion obtained from Echinodorus grandiflorus leaves has effective diuretic, natriuretic and hypotensive activity on rats after acute treatment. It is known that drugs with diuretic action are considered as first choice for the treatment of hypertension, especially by increasing urine flow and renal sodium excretion, as well as their important vasodilator effects (McManus et al., 2012). Currently, the pharmaceutical market offers several treatment options of diuretic drugs, especially loop diuretics and thiazides. Despite their high effectiveness, adverse effects caused by continued use or high doses of these drugs have led to a low adherence to treatment. The main cause of this problem may be explained at least in part by increased urinary elimination of electrolytes, leading to the onset of metabolic and cardiac disorders, including metabolic acidosis and arrhythmias. Moreover, the prolonged action of diuretics hinders the activity of compensatory mechanisms responsible for the reabsorption of ions, generating mostly an electrolyte imbalance (Ellison and Loffing, 2009).

In developing this study, we propose to investigate effective therapeutic alternatives based on ethnopharmacological validation of various natural products. Thus, if the test substance presents a significant diuretic and effective natriuretic effect associated with a small potassium and bicarbonate excretion, it could become a promising therapeutic option with low probability of causing adverse effects. To our surprise, two species widely used as diuretic in Brazil showed no diuretic properties and do not justify its popular use in the way they have been used for decades. On the other hand, the infusion obtained from Echinodorus grandiflorus showed acute diuretic effect, similar to that obtained with hydrochlorothiazide, a widely used thiazide diuretic. In this study, hydrochlorothiazide at a dose of 25 mg/kg showed high effectiveness, but eliminated large amounts of potassium and bicarbonate in the urine. Currently, it is known that thiazides exert their diuretic effect through inhibition of the Na +/Cl- cotransporter in the tubular distal convoluted, causing a significant retention of NaCl in the renal tubules that provides a large excretion of water, chloride, sodium and potassium. Moreover, some members of this group retain significant carbonic anhydrase inhibitory activity (Jackson, 1996). Although the effects of ES-EG on urine volume, sodium, potassium, and chloride excretion was similar to that obtained with hydrochlorothiazide, a significant difference in relation to bicarbonate excretion was observed, showing a significant sparing effect of this ion. Although the results obtained are very significant, it remains unclear through which mechanisms ES-EG exerts its diuretic effects. In recent years, several studies have been published about the pharmacologic mechanisms that form the basis of the diuretic properties of various natural products. Many medicinal plants and their secondary metabolites such as flavonoids and condensed tannins can cause diuretics effects through several mechanisms, including ACE inhibition and bradykinin receptor

activation with subsequent release of endothelial nitric oxide and prostaglandins (Gasparotto Junior et al, 2009; 2012;Leme et al, 2013). Moreover, some effects on vasopressin release (Kazama et al., 2012) and osmotic properties have been described, primarily due to the occurrence of large amounts of metals in the parenchyma of several herbal medicines (Benjumea et al., 2008). It is too premature for us to suggest any mechanism of action of ES-EG, and other in-depth studies should be conducted for this purpose. On the other hand, the large electrolyte excretion induced by species is not characteristic of drugs that inhibit the release of vasopressin, and the small amount of metal naturally found in Echinodorus grandiflorus leaves (data not shown) can exclude a probable osmotic effect. Furthermore, although other studies have shown “in vitro” ACE inhibitor effects of different extract obtained from these species, no “in vivo” effect on this enzyme were observed in this study, excluding a probable effect related to this mechanism. When the diuretic effects induced by ES-EG are observed, it was found that the major components found in this infusion and that could be the agents responsible for this activity are several flavonoids C-glycosides including swertisin, isovitexin, isoorientin, swertiajaponin, and their diglycoside derivatives. In recent years, studies have shown the role played by flavonoids in reducing and preventing the formation of free radicals and other oxidizing agents. In addition, these compounds were closely related with diuretic, antihypertensive and antiatherosclerotic activity of several natural products (Gasparotto Junior et al., 2011; 2012; Brant et al, 2014). As phenolic compounds are classical antioxidant agents, and this effect could increase the bioavailability of endothelial nitric oxide leading to dilatation of the renal afferent arterioles and increasing glomerular filtration rate (Gasparotto Junior et al., 2012), we chose to perform an initial screening using three classic “in vitro” antioxidant

tests. So, all infusions tested (ES-EG, ES-CC, and ES-PT) were able to promote a strong (“in vitro”) antioxidant activity in the three models used. On the other hand, only ES-EG was able to increase the bioavailability of endothelial nitric oxide (measured by serum nitrate), with possible effects in systemic and renal vasculature. In fact, an important increase in renal cortical blood flow was observed, which could influence the glomerular filtration rate and induce diuresis. Moreover, as previously reported with another extraction process and different administration route (Lessa et al., 2008), a significantly acute hypotensive effect was also observed, without affecting heart rate after intraduodenal administration. Further pharmacological studies should be carried out in order to investigate the molecular mechanisms involved in diuretic and hypotensive effects of ES-EG and understand the real role of nitric oxide and antioxidant properties of this extract in renal and hemodynamic effects. In summary, the results presented here shown that the ES-EG obtained from Echinodorus grandiflorus leaves shown a significant diuretic and hypotensive activity and suggest that these effects could be related with an important renal and systemic vasodilator effect. In addition, it was shown for the first time that the pharmacological effects of ethanol soluble fractions obtained from Phyllanthus tenellus and Cuphea carthagenensis do not support their popular use as a diuretic agent. However, since the isolated compounds of these species are potentially promising as pharmacological agents, future studies should elucidate the probable pharmacological mechanisms culturally assigned to these plants.

Acknowledgements

We are grateful to Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT-Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil), Diretoria Executiva de Gestão da Pesquisa e Pós-Graduação (DEGPP/UNIPAR-Brazil) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil), especially to ProEquipment Program, for financial support. All authors declare no financial/commercial conflicts of interest regarding the information in this article.

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Legend to figures

Figure 1. UPLC analysis of ES obtained from Echinodorus grandiflorus (A), Phyllanthus tenellus (B), and Cuphea carthagenensis (C).

Figure 2. ES obtained from Echinodorus grandiflorus induced hypotension (D-F) and increase renal cortical blood flow (A-C) in normotensive rats. The animals were subjected to an intraduodenal treatment with ES-EG (30-300 mg/kg), ES-CC (30-300 mg/kg), ES-PT (30-300 mg/kg), or vehicle. The hypotensive and renal cortical blood flow effects were measured for 45 minutes after administration of distilled water (closed bars) or extracts (open bars). The results are the mean ± S.E.M. of five animals in each group. The statistical evaluation was carried out by analysis of variance (ANOVA) followed by a

Dunnett's test. p < 0.05 when compared with the respective control group.

Figure 3. ES obtained from Echinodorus grandiflorus protects against lipid peroxidation “in vitro” (A-C) and increases serum nitrate levels (D-F) in Wistar rats. The serum was obtained after 8 hours of treatment. The results are the mean ± S.E.M. of five animals in each group or “in vitro” experiments performed in triplicate. The statistical evaluation was carried out by analysis of variance (ANOVA) a

followed by Dunnett's test. p < 0.05 when compared with the respective control group (D) or negative b

control group (A-C) (erythrocytes with saline). p < 0.05 when compared with the positive control group (A-C) (erythrocytes with AAPH).

FIGURE 1 Prando et al.

FIGURE 2 Prando et al.

FIGURE 3 Prando et al.

Graphical abstract

Table 1. Effect of acute oral administration of ethanol soluble fractions (ES) obtained from Echinodorus grandiflorus (ES-EG), Phyllanthus tenellus (ES-PT) and Cuphea carthagenensis (ES-CC) on urinary volume, pH, conductivity and density

Group Control HCTZ (25 mg/kg) ES-EG (30 mg/kg) ES-EG (100 mg/kg) ES-EG (300 mg/kg) ES-PT (30 mg/kg) ES-PT (100 mg/kg) ES-PT (300 mg/kg) ES-CC (30 mg/kg) ES-CC (100 mg/kg) ES-CC (300 mg/kg)

Urine volume (8h/100g) 3.2 ± 0.3

6.6 ± 0.2

Conductivity (mS/cm) 15.4 ± 0.2

a

6.6 ± 0.4

6.7 ± 0.1

17.2 ± 0.1a

1009 ± 0.6

4.4 ± 0.2

6.2 ± 0.2

15.2 ± 0.1

1013 ± 0.6

4.6 ± 0.2a

6.4 ± 0.3

15.9 ± 0.1

1013 ± 0.7

5.4 ± 0.3a

6.3 ± 0.3

16.8 ± 0.2a

1012 ± 1.4

3.0 ± 0.2

6.4 ± 0.1

14.6 ± 0.1

1010 ± 1.2

3.2 ± 0.2

6.4 ± 0.2

15.5 ± 0.2

1011 ± 1.2

3.1 ± 0.3

7.1 ± 0.2

15.0 ± 0.2

1012 ± 1.0

4.1 ± 0.7

6.6 ± 0.2

15.1 ± 0.2

1012 ± 1.3

3.5 ± 0.6

6.9 ± 0.1

14.9 ± 0.3

1010 ± 1.2

3.8 ± 0.6

6.7 ± 0.1

15.3 ± 0.3

1011 ± 1.3

pH

Density (g/mL) 1013 ± 0.3

Values are expressed as mean ± S. E. M. of five rats in each group in comparison with the control using one-way ANOVA followed by Dunnet test (ap < 0.05).

Table 2. Effect of acute oral administration of ethanol soluble fractions (ES) obtained from Echinodorus grandiflorus (ES-EG), Phyllanthus tenellus (ES-PT) and Cuphea carthagenensis (ES-CC) on urinary electrolyte excretion

Saluretic indexb

ElNa+ (µEq/min/1 00g)

Elk+ (µEq/min/1 00g)

ElCl(µEq/min/1 00g)

ElHCO3(µEq/min/1 00g)

Na

0.76 ± 0,07

0.27 ± 0,02

1.51 ± 0.21

0.12 ± 0.03

Z (10

1.73 ±

0.63 ±

mg/k

0.14a

0.08a

3.95 ± 0.24a

0.32 ± 0.02a

0.69 ± 0.09

0.25 ± 0.03

2.26 ± 0.16a

0.15 ± 0.02

0.24 ± 0.02

2.39 ± 0.16a

0.18 ± 0.03

0.19 ± 0.03

Grou p Cont rol

K+

Cl-

-

-

-

2.2

2.3

2.6

7

3

1

0.9

0.9

1.4

0

2

9

1.7

0.8

1.5

1

8

8

1.8

1.5

1.8

6

9

9

1.0

0.9

0.8

6

2

6

1.1

1.0

0.9

8

7

8

+

HC O3-

HCT 2.66

g) ESEG (30 mg/k

1.25

g) ESEG (100 mg/k

1.30 ± 0,06a

1.50

g) ESEG (300 mg/k

1.42 ±

0.43 ±

0,07

0.03

2.86 ± 0.24a

0.81 ± 0.06

0.25 ± 0.03

1.30 ± 0.12

0.13 ± 0.01

0.90 ± 0.09

0.29 ± 0.02

1.42 ± 0.06

0.14 ± 0.01

a

a

1.58

g) ESPT (30 mg/k

1.08

g) ESPT (100

1.16

mg/k g) ESPT (300

0.86 ± 0.08

0.31 ± 0.04

1.27 ± 0.14

0.13 ± 0.01

0.78 ± 0.06

0.28 ± 0.03

1.38 ± 0.20

0.18 ± 0.04

0.79 ± 0.08

0.30 ± 0.02

1.45 ± 0.06

0.19 ± 0.02

0.82 ± 0.05

0.28 ± 0.02

1.17 ± 0.17

0.17 ± 0.03

mg/k

1.1

1.1

0.8

3

4

4

1.0

1.0

0.9

2

3

1

1.0

1.1

0.9

3

1

6

1.0

1.0

0.7

7

3

6

1.08

g) ESCC (30 mg/k

1.50

g) ESCC (100 mg/k

1.58

g) ESCC (300 mg/k

1.41

g) Values are expressed as mean ± S. E. M. of five rats in each group in comparison with the control using one-way ANOVA followed by Dunnet test (ap < 0.05). bSaluretic index = mmol/L problem group/mmol/L control group. El: Excreted load; HCTZ: hydrochlorothiazide.

Table 3. IC50 and maximum activity (%) of DPPH free radical scavenging and nitric oxide (NO) radical scavenging of standard antioxidants and the ethanol soluble fractions (ES) from Echinodorus grandiflorus (ES-EG), Phyllanthus tenellus (ES-PT) and Cuphea carthagenensis (ES-CC).

DPPH free radical scavenging IC50 (μg/mL)

%

μg/mL

NO radical scavenging IC50 (μg/mL)

%

μg/mL

ES-EG

102 ± 4.3

90 ± 2.6

300

98 ± 2.1

90 ± 3.5

300

ES-CC

18 ± 4.1

95 ± 1.8

30

465 ± 4.1

68 ± 2.5

1000

ES-PT

6 ± 2.9

96 ± 2.1

100

512 ± 2.9

65 ± 2.1

1000

42 ± 0.7

95 ± 5.3

100

Ascorbic acid

2.0 ± 0.3 97 ± 3.1

10

Values are expressed as mean ± S. E. M. of triplicate experiments.