Mechanisms underlying the diuretic effects of Tropaeolum majus L. extracts and its main component isoquercitrin

Journal of Ethnopharmacology 141 (2012) 501–509

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Mechanisms underlying the diuretic effects of Tropaeolum majus L. extracts and its main component isoquercitrin Arquimedes Gasparotto Junior a,∗ , Thiago Bruno Lima Prando a , Thiago dos Santos Vilhena Leme a , Francielly Mourão Gasparotto a , Emerson Luiz Botelho Lourenc¸o a,b , Yanna Dantas Rattmann c , José Eduardo Da Silva-Santos d , Cândida Aparecida Leite Kassuya e , Maria Consuelo Andrade Marques b a

Institute of Biological Sciences, Medical and Health, Universidade Paranaense, Umuarama, PR, Brazil Department of Pharmacology, Universidade Federal do Paraná, Curitiba, PR, Brazil c Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Curitiba, PR, Brazil d Department of Pharmacology, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil e Faculty of Medical Sciences, Universidade Federal da Grande Dourados, Dourados, MS, Brazil b

a r t i c l e

i n f o

Article history: Received 13 November 2011 Received in revised form 10 March 2012 Accepted 12 March 2012 Available online 21 March 2012 Keywords: Tropaeolum majus L. ACE Na+ /K+ /ATPase Isoquercitrin

a b s t r a c t Ethnopharmacological relevance: Previous studies have shown that the extracts obtained from Tropaeolum majus L., and its main compound isoquercitrin (ISQ), exhibit pronounced diuretic effects, supporting the ethnopharmacological use of this plant. The aim of this study was to evaluate the efficacy and mechanisms underlying the diuretic action of an ethanolic extract of Tropaeolum majus (HETM), its purified fraction (TMLR), and its main compound ISQ, in spontaneously hypertensive rats (SHR). Materials and methods: The diuretic effects of HETM (300 mg/kg; p.o.), TMLR (100 mg/kg; p.o.), and ISQ (10 mg/kg; p.o.), were compared with classical diuretics in 7 days repeated-dose treatment. The urinary volume, sodium, potassium, chloride, bicarbonate, conductivity, pH and density were estimated in the sample collected for 15 h. The plasmatic concentration of sodium, potassium, urea, creatinine, aldosterone, vasopressin, nitrite and angiotensin converting enzyme (ACE) activity were measured in samples collected at the end of the experiment (seventh day). Using pharmacological antagonists or inhibitors, we determine the involvement of bradykinin, prostaglandin and nitric oxide (NO) in ISQ-induced diuresis. In addition, reactive oxygen species (ROS) and the activity of erythrocytary carbonic anhydrase and renal Na+ /K+ /ATPase were evaluated in vitro. Results: HETM, TMLR and ISQ increased diuresis similarly to spironolactone and also presented K+ -sparing effects. All groups presented both plasmatic aldosterone levels and ACE activity reduced. Previous treatment with HOE-140 (a B2-bradykinin receptor antagonist), or indomethacin (a cyclooxygenase inhibitor), or L-NAME (a NO synthase inhibitor), fully avoided the diuretic effect of ISQ. In addition, the 7 days treatment with ISQ resulted in increased plasmatic levels of nitrite and reducing ROS production. Moreover, the renal Na+ /K+ /ATPase activity was significantly decreased by ISQ. Conclusion: Our results suggest that the mechanisms through ISQ and extracts of Tropaeolum majus increase diuresis in SHR rats are mainly related to ACE inhibition, increased bioavailability of bradykinin, PGI2, and nitric oxide, besides an inhibitory effect on Na+ /K+ -ATPase. © 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Diuretics, such as thiazides and furosemide, are among the most used medicines to treat cardiovascular diseases in humans. These

∗ Corresponding author at: Laboratory of Cardiovascular and Renal Pharmacology, Universidade Paranaense – Prac¸a Mascarenhas de Moraes s/n – Centro, Caixa Postal 224, Umuarama – PR 87.502-210, Brazil. Tel.: +55 44 3621 2828; fax: +55 44 3621 2830. E-mail address: [email protected] (A. Gasparotto Junior). 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.03.018

drugs are well known by their ability to reduce blood pressure in hypertension and improve the cardiovascular function in heart failure. However, diuretics are also associated with several adverse effects, such as changes in systemic electrolyte levels (hypokalemia, hyperkalemia and hyponatremia), hypovolemia, metabolic alkalosis or acidosis, and hyperuricemia, among others (Jackson, 1996). Thus, it makes sense that the development of new diuretic agents may not only improve the output in several cardiovascular diseases, may also reduce the incidence of undesired effects. In recent years, several reports studying natural products have focused on basic and clinical research for new diuretic drugs. These

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reports are predominantly supported by empirical usage (Wright et al., 2007; Tovchiga and Shtrygol, 2009), and although most of them have presented significant effects in animal models, their mechanisms of action have been scarcely investigated. Previous studies have shown that an ethanolic extract (HETM), and their purified fraction (TMLR), obtained of leaves of Tropaeolum majus L. induce dose-dependent diuresis in rats, explaining, at least in part, its popular usage against cardiovascular diseases. This activity is directly related to the presence of the flavonoid isoquercitrin (ISQ), and shows saluretic/diuretic effects interestingly associated with potassium-saving activity in spontaneously hypertensive rats (SHR) (Gasparotto Junior et al., 2009, 2011a). Taking into account the popular usage and previous evidence of efficacy and the therapeutic potential of Tropaeolum majus against cardiovascular disorders, this study was carried out in order to provide mechanistically relevant effects that may underlie the diuretic effects induced by preparations obtained from Tropaeolum majus, as well as its main compound isoquercitrin, in spontaneously hypertensive rats (SHR). 2. Materials and methods 2.1. Drugs Isoquercitrin (ISQ), furosemide, hydrochlorothiazide, acetazolamide, spironolactone, ouabain octahydrate, N-␻-nitro-l-arginine methyl ester (l-NAME), apocynin, N-hippuryl-l-histidyl-l-leucine hydrate, o-phthalaldehyde, Icatibant acetate (HOE-140), 2 ,7 dichlorofluorescein-diacetate (DCFH-DA), indomethacin, and captopril, were obtained from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA). All other drugs and reagents used were purchased from Merck (Darmstadt, Germany). 2.2. Phytochemical study 2.2.1. Plant material and preparation of the HETM and TMLR The leaves of Tropaeolum majus were collected in June 2008 at the botanical garden of Universidade Paranaense (UNIPAR) (Umuarama, Brazil), which is located at 430 m altitude above sea level (S23◦ 47 55–W53◦ 18 48). The plant was identified by Dr. Mariza Barion Romagnolo (Department of Botany, UNIPAR, Brazil). A voucher specimen is deposited at the Herbarium of this University under number 2230. The leaves were air-dried in an oven at 40 ◦ C for 4 days, and then cut and pulverized. The resulting dried powdered plant material was macerated for 7 days in 90% ethanol solution. The solvent was removed using a rotary vacuum evaporator under reduced pressure and lyophilized, giving up 15.39% of the dry material extracted (HETM). This extract was suspended in water, filtered and subjected to a column with Amberlite XAD-2, eluted with water and ethanol. The fraction eluted with ethanol (TMLR) yielded 15.30%, after solvent evaporation. The chemical composition of TMLR was previously described (Gasparotto Junior et al., 2011b). 2.2.2. Atomic absorption spectrophotometry (AAS) For measurement of metals content (chromium, nickel, lead, iron, zinc, copper, sodium, potassium, calcium and magnesium) from the leaves of Tropaeolum majus we added 6 mL of nitric acid and 3 mL of hydrogen peroxide (30%) in 0.5 g of sample (dried and crushed leaves). The mixture was placed on a heating plate at 100 ◦ C for 90 min, filtered and packaged in a flask, completed to 50 mL with Milli-Q water (Fraige et al., 2007). The digestions were performed in triplicate. The reliability of the process of digestion was verified by recovery of all elements, which ranged from 90 to 102%. The concentration of metals in the samples was performed using AAS and calculated by linear regression using calibration curves with

at least seven points for each element. The regression coefficient obtained in these experiments were greater than 0.997. 2.3. Pharmacological studies 2.3.1. Animals We used male spontaneously hypertensive rats (SHR) (3–4 months old, weighing between 250 and 300 g) from the colony of the Institute of Biomedical Science of Universidade de São Paulo (USP, São Paulo, Brazil). All animals were maintained under standard laboratory conditions, with a constant 12 h light/dark cycle and controlled temperature (22 ± 2 ◦ C). Standard pellet food (Nuvital® , Curitiba/PR, Brazil) and water were available ad libitum. All experimental procedures adopted in this study were previously approved by the Institutional Ethics Committee of the Universidade Federal do Paraná (UFPR, Brazil; authorization number 240). 2.3.2. Assessment of diuretic activity: experimental design The diuretic activity was determined accordingly to previous descriptions (Kau et al., 1984), with minor modifications. The animals were separated in eight groups (n = 5) along with the 7 days repeated-dose study. Rats were fasted overnight with free access to water and subjected to treatment as subsequently described. Each animal was placed in an individual metabolic cage 24 h before the commencement of the treatments for environmental adaptation. 2.3.2.1. Comparative studies with classical diuretics in 7 days repeated-dose treatment. HETM (300 mg/kg), TMLR (100 mg/kg), ISQ (10 mg/kg), hydrochlorothiazide (HCTZ; 10 mg/kg), furosemide (FURO; 10 mg/kg), acetazolamide (ACTZ; 10 mg/kg), or spironolactone (SPIRO; 50 mg/kg), were administered once daily to separate groups by oral route (gavage) for 7 days. The control group received vehicle only. In the seventh day the urine was collected in a graduated cylinder and its volume was measured for 15 h. Cumulative urine excretion was calculated in relation to body weight and expressed as mL/100 g. Electrolyte concentrations (Na+ , K+ , Cl− and HCO3 − ), pH, density, and conductivity were estimated from urine sample of each rat. The plasmatic concentration of sodium, potassium, urea, creatinine, aldosterone, and vasopressin, were also measured at the end of the experiment (7th day). 2.3.2.2. Analytical procedures. For serum analysis, blood samples were collected in conical tubes after decapitation. Plasma and serum were obtained by centrifugation (800 × g, 10 min, 4 ◦ C) and stored at −20 ◦ C until their analysis. The urinary and plasmatic levels of sodium and potassium were quantified by flame photometry. The chloride and bicarbonate concentrations were quantified by argentimetry (titration). The urea and creatinine were determined by enzymatic method using an automated analyzer BM/Hitachi 912 (Cobas Mira, Roche, Indianapolis, USA). Serum vasopressin was measured by radioimmunoassay. Aldosterone levels were estimated by enzyme linked immunosorbent assay (ELISA, Immuno-Biological Laboratories, Inc.) (Al-Dujaili et al., 2009). 2.3.3. Evaluation of the mechanisms involved in the diuretic activity 2.3.3.1. In vitro determination of renal Na+ /K+ /ATPase activity. The Na+ /K+ /ATPase activity was determined by the Noel and Godfraind (1984) method with slight modifications (Pocas et al., 2003). The specific activity of the enzyme corresponds to the difference between the total ATPase activity and the activity measured in the presence of 1 mM ouabain (ouabain-resistant activity). The quantity of protein was adjusted in order to hydrolyze no more than 10–15% of the substrate during the incubation period. The reaction was started by addition of the kidney samples, incubated at 37 ◦ C for

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2 h, in a total volume of 0.5 mL. The incubation was performed in the presence of 84 mM NaCl, 3 mM KCl, 3 mM MgCl2 , 1.2 mM ATPNa2 , 2.5 mM EGTA, 10 mM sodium azide and 20 mM maleic acid buffered to pH 7.4 with Tris. Inhibition curves were obtained in the presence of increasing concentrations of HETM, TMLR or ISQ (3–30 ␮M). The extract concentration was estimated based on the amount of isoquercitrin (molecular weight, 464.38) present in HETM and TMLR (Gasparotto Junior et al., 2011b). 2.3.3.2. Measurement of erythrocyte carbonic anhydrase activity. After the 7 days of treatment with vehicle, HETM (300 mg/kg), TMLR (100 mg/kg), ISQ (10 mg/kg), or acetazolamide (10 mg/kg), blood samples were collected from SHR rats. One mL of red blood cells was added to 3 mL of distilled water followed by 3 ml of chloroform, and subsequent centrifugation at 8000 × g for 10 min. The supernatant was collected and diluted in the ratio 1:10 (v/v) in distilled water, resulting in a hemolysate at a dilution of 1/40 (v/v). Two samples of hemolysate (50 ␮l) were incubated with alphanaphthyl acetate (1 mL, 1.7 mM) in phosphate buffer (0.02 M, pH 7.0) containing dioxane (2%). The first sample was incubated with acetazolamide (1 mL at 8 mM in NaOH) at 37 ◦ C for 20 min. The second sample was exposed to acetazolamide at the end of the incubation period (37 ◦ C for 20 min). Then, 500 ␮l of 5-chloro-otoluidine (2 mg/ml) was added to the samples and, after 15 min, the absorbance was measured at 555 nm. Each 1 mmol of acetate alpha-naphthol produced per minute per ml of hemolysate was considered as one unit (U) (Tashian, 1989). 2.3.3.3. Angiotensin converting enzyme (ACE) assay. ACE activity was determined in serum samples obtained from SHR rats after the 7 days of treatment with HETM (300 mg/kg), TMLR (100 mg/kg), ISQ (10 mg/kg), or vehicle (control group), as previously described (Santos et al., 1985). Blood was collected into glass tubes after decapitation and centrifuged for serum separation. The serum (10 ␮l) was incubated for 15 min at 37 ◦ C with 490 ␮l of assay solution (composition: NaCl 0.9 M and Hip-His-Leu at 5 mM in 0.4 M sodium borate buffer, pH 8.3) The reaction was stopped by addition of 1.2 mL of NaOH (0.34 N). The production of HisLeu was measured fluorometrically (365 nm excitation and 495 emission, Aminco Model J4-7461 fluoromonitor, American Instrument Co., Silver Springs, MD) after the addition of 100 ␮l of o-phthaldialdehyde (20 mg/ml in methanol), and 200 ␮l of HCl (3 N), followed by centrifugation (800 × g, 5 min) at room temperature. To correct intrinsic fluorescence of plasma, time-zero blank samples were prepared by adding plasma after NaOH treatment. In some experiments, captopril (60 mg/kg) was used as positive control. All measurements were made in triplicate. 2.3.3.4. Study of the involvement of bradykinin, prostaglandins and nitric oxide in the diuretic activity. This study was performed accordingly to previous descriptions (Gasparotto Junior et al., 2009) with minor modifications. After 6 days of treatment (thus, in the morning of the seventh day), different groups of SHR rats received HOE140 (a B2 bradykinin receptor antagonist; 1.5 mg/kg, given 15 min before; i.p.), indomethacin (a cyclooxygenase inhibitor; 5 mg/kg, given 1 h before; p.o.) or l-NAME (a nitric oxide synthase inhibitor; 60 mg/kg, given 1 h before the experiments; p.o.), followed by oral administration of deionized water (5 ml/kg, control group), HETM (300 mg/kg), TMLR (100 mg/kg), ISQ (10 mg/kg), or spironolactone (50 mg/kg). The urine volume, as well as its content of sodium, was measured 15 h after these treatments. 2.3.3.5. Determination of nitrate/nitrite serum (NOx). The plasma nitrite concentration was determined by reducing nitrate enzymatically using the enzyme nitrate reductase, as previously described

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(Schmidt et al., 1989). After the 7 days of treatment with vehicle, ISQ (5 and 10 mg/kg), or apocynin (NADPH oxidase inhibitor, 100 mg/kg), plasma samples collected from SHR rats were deproteinized with zinc sulfate (30 mmol) and diluted 1:1 with Milli-Q water. For the conversion of nitrate to nitrite, samples were incubated at 37 ◦ C for 2 h in the presence of nitrate reductase expressed in Escherichia coli. After the incubation period, samples were centrifuged (800 × g, 10 min) to remove the bacteria. Then, 100 ␮l of supernatant was 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 curves of nitrite and nitrate (0–150 mM) were performed simultaneously. 2.3.3.6. Reactive oxygen species (ROS) generation in blood vessels from SHR treated rats. To evaluate the production of ROS in mesenteric arteries from SHR rats treated for 7 days with ISQ (5 and 10 mg/kg), or apocynin (100 mg/kg), samples of the superior mesenteric artery were obtained and frozen in a mounting medium for fresh tissue (Tissue-Tek® OCT, Optimal Cutting Temperature), and stored at −80 ◦ C. Cross sections (25 mm thick) from frozen segments in Tissue-Tek® were obtained in a cryostat microtome at −22 ◦ C. The slides were then placed at room temperature in a moist chamber (20 min). The production of ROS in vascular samples was assessed using the probe 2 ,7 -dichlorofluorescein-diacetate (DCFH-DA), a lipid-permeable non-fluorescent compound that can be oxidized by intracellular ROS to the fluorescent compound 2 ,7 dichlorofluorescein (DCF), which is lipid-impermeable (Royall and Ischiropoulos, 1993). This probe was incubated for 30 min at 37 ◦ C. After these procedures, the slides were set in a mounting medium anti-laundering (Dako® ), and examined under confocal microscopy (Confocal Radiance 2100; Bio Rad, Hercules, CA, USA) with a 20× objective. The excitation of samples was performed at 488 nm with an argon laser and the emission signal was recorded through a 565–610 nm filter. The program Sharp 2000 (Bio-Rad Microscience Ltd.) was used to quantify the fluorescence. 2.4. Statistical analysis The results are expressed as mean ± standard error of mean (S.E.M) of 5 animals per group. Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s test or Student’s t-test, when applicable. A p value less than 0.05 were considered statistically significant. The graphs were drawn and the statistical analyses were performed using GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA).

3. Results 3.1. Metals content in the leaves of Tropaeolum majus In order to determine if the diuretic effect of Tropaeolum majus extracts (HETM and TMLR) could be related to osmotic effects, caused by large amounts of potassium and/or other metals found in this specie, we carried out an chemical analyses by AAS to evaluate the content of metals present in the leaves of this plant. The results revealed very low amounts of potassium and/or other metals in Tropaeolum majus leaves (data no shown), in agreement with quality standards established by the National Agency for Sanitary Surveillance (ANVISA) for food and beverages (Brasil, 1998).

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3.2. Comparative studies with reference diuretics The oral administration of all drugs tested (HETM, TMLR and ISQ), including classical diuretics, resulted in increased diuresis when compared to control group (Fig. 1). The urinary output measured in HETM, TMLR and ISQ groups were closed to that found in ACTZ, SPIRO and FURO groups and slightly lesser than in HCTZ group (Fig. 1A). Likewise, HCTZ treated animals presented higher amounts of sodium in their urine, when compared to Tropaeolum majus extracts (Fig. 1B). Both ACTZ and HCTZ groups presented increased urinary excretion of potassium (by 72 ± 11% and 88 ± 18%, respectively, when compared to control group), a parameter that remained unchanged in animals treated with Tropaeolum majus extracts (HETM and TMLR) and ISQ groups. In those animals treated with ACTZ, the pH and urinary concentration of bicarbonate were also statistically different than in control group (Table 1). In addition, the urinary excretion of chloride was increased by 135 ± 17% and 92 ± 12% in FURO and HCTZ groups, respectively, when compared to control rats. The density and the urinary conductivity did not present significant differences among the groups (Table 1). Since the general profile of diuretic effects displayed by Tropaeolum majus extracts and ISQ were closed to the one induced by spironolactone, the following experiments were performed using this diuretic drug for comparison.

3.3. Effect on aldosterone, vasopressin, urea, creatinine and electrolyte levels in plasma The effects of 7 days of treatment with HETM, TMLR and ISQ on serum aldosterone e vasopressin are presented in Fig. 2B and C. Administration of TMLR and ISQ reduced aldosterone in SHR rats, when compared to the control group (treated with vehicle only) by 14 ± 3% and 12 ± 2%, respectively. None of the substances tested induced similar effects in vasopressin levels. The plasmatic amounts of creatinine, urea, sodium, and potassium in plasma, measured on the final day of the treatment, have not been impaired in animals subjected to our experimental protocols (data no shown).

3.4. Inhibitory effects of the Tropaeolum majus extracts (HETM and TMLR) and ISQ (ISQ) on ACE activity The plasmatic ACE activity after treatment with HETM (300 mg/kg) was reduced by 32 ± 7%. Moreover, the treatment with TMLR (100 mg/kg) and ISQ (10 mg/kg) reduced ACE activity by 38 ± 6% and 43 ± 8%, respectively. As expected, the positive control captopril (60 mg/kg) reduced the ACE activity around 65% (Fig. 2A).

3.5. Erythrocyte carbonic anhydrase activity The treatment with all tested drugs (HETM, TMLR or ISQ) did not change the erythrocyte carbonic anhydrase activity, while the administration of acetazolamide, a classic carbonic anhydrase inhibitor reduced it by 44 ± 6% (data no shown).

3.6. Inhibitory effects of Tropaeolum majus extracts and ISQ on renal Na+/K+/ATPase activity Incubation of HETM, as well as TMLR or ISQ (30 ␮M) in kidney samples obtained from SHR rats reduced the activity of Na+ /K+ /ATPase by 12 ± 2%, 14 ± 4%, and 17 ± 3%, respectively (Fig. 3A–C).

3.7. Acute administration of HOE-140 reduces the diuretic response of HETM, TMLR and ISQ after 7 days of treatment The pre-treatment with HOE-140 (a B2 bradykinin receptor antagonist) decreased by 49 ± 3%, 34 ± 6% and 33 ± 4% the urinary volume excreted by animals treated with HETM, TMLR and ISQ, respectively (Fig. 4A). Likewise, urinary sodium excretion was reduced by 17 ± 4% in those animals that received HETM, 26 ± 4% and 16 ± 3% in the groups treated with TMLR and ISQ, respectively (Fig. 4B). On the other hand, the diuretic effect of spironolactone remained unchanged after treatment with HOE-140 (Fig. 4A and B). 3.8. Acute administration of indomethacin abolishes the diuretic response of HETM, TMLR and ISQ after 7 days of treatment The treatment with indomethacin (1 h before the experiments) reduced the diuresis by 48 ± 5%, when compared with the group that received HETM only. Similarly, SHR rats treated with TMLR and ISQ had their diuresis reduced by 38 ± 9% and 34 ± 7%, respectively (Fig. 5A). In addition, the urinary excretion of sodium was reduced by 26 ± 4% in the group treated with HETM, and by 28 ± 6%, and 23 ± 4%, in TMLR and ISQ treated animals, respectively (Fig. 5B). The diuretic effect of spironolactone remained unchanged after the treatment with indomethacin (Fig. 5A and B). 3.9. Effects of acute administration of l-NAME on urinary volume and sodium concentration after 7 days of treatment with HETM, TMLR and ISQ The pre-treatment with l-NAME decreased by 30 ± 9%, 35 ± 13% and 33 ± 11% the urine volume excreted by animals that received HETM, TMLR and ISQ, respectively (Fig. 6A). In addition, the urinary excretion of sodium was reduced by 23 ± 9%, 20 ± 11%, and 24 ± 8% in HETM, TMLR and ISQ groups, respectively (Fig. 6B). The diuretic effect of spironolactone remained unchanged after the treatment with l-NAME (Fig. 6A and B). 3.10. Increase of serum nitrite + nitrate in SHR rats after 7 days of treatment with ISQ The daily treatment with ISQ (10 mg/kg), but not apocynin, significantly increased the serum nitrate + nitrite levels when compared to the control group (ISQ 143 ± 21 ␮M; control 51 ± 6 ␮M) (Fig. 7A). 3.11. ISQ reduces reactive oxygen species (ROS) in mesenteric arteries of SHR rats The results obtained from identification of ROS by confocal microscopy are presented in Fig. 7 (pictures and panel B). The amounts of ROS were reduced by 62 ± 4%, 52 ± 11%, and 20 ± 8% in samples from SHR rats treated with ISQ (5 and 10 mg/kg), and apocynin (APC; 100 mg/kg), respectively (Fig. 7B). 4. Discussion and conclusions Tropaeolum majus is widely distributed and used as a popular medicine around the world. Its traditional application includes the treatment of cardiovascular and renal disorders (Panizza, 1998; Lorenzi and Matos, 2002). Nevertheless, the cardiovascular effects of extracts obtained from Tropaeolum majus had been scarcely investigated. In previous studies we have demonstrated that the infusion and a crude extract (HETM) prepared from leaves of Tropaeolum majus (Gasparotto Junior et al., 2009) were able to cause diuretic effects in a dose-dependent manner when orally

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Fig. 1. Comparative effects of the treatment for 7 days with Tropaeolum majus extracts (HETM and TMLR), isoquercitrin (ISQ), acetazolamide (ACTZ), spironolactone (SPIRO), furosemide (FURO) and hydrochlorothiazide (HCTZ) on volume (A) and sodium urinary (B). The urine samples were collected for 15 h in the seventh day. Each bar represents the mean of five animals (SHR rats) and the vertical lines show the S.E.M. Asterisks or # denote the significance levels in comparison with the control group and HETM, TMLR or ISQ treated rats, respectively (one-way ANOVA followed by Bonferroni test) (#, *p < 0.05; **p < 0.01).

Table 1 Comparative effects of 7 days of treatment with Tropaeolum majus extracts (HETM and TMLR), isoquercitrin (ISQ), acetazolamide (ACTZ), spironolactone (SPIRO), furosemide (FURO), or hydrochlorothiazide (HCTZ), on urinary electrolyte excretion, conductivity, pH and density. Group

n

K+ (mmol/l/15 h)

Control HETM (300 mg/kg) TMLR (100 mg/kg) ISQ (10 mg/kg) ACTZ (10 mg/kg) SPIRO (50 mg/kg) FURO (10 mg/kg) HCTZ (10 mg/kg)

5 5 5 5 5 5 5 5

54.38 52.25 41.13 39.75 90.88 36.88 58.63 97.13

± ± ± ± ± ± ± ±

4.41 5.42 5.60 5.21 5.11a,b 5.78 4.60 6.81a,b

HCO3 − (mmol/l/15 h)

Cl− (mmol/l/15 h) 10.31 11.20 12.35 11.55 14.75 12.48 26.33 20.17

± ± ± ± ± ± ± ±

1.22 1.34 1.21 1.26 1.87 1.32 2.17a,b 2.08a,b

120.10 125.22 129.53 117.8 311.74 118.22 136.84 158.25

± ± ± ± ± ± ± ±

6.81 7.35 6.35 6.68 22.42a,b 9.16 14.59 16.33

pH 6.09 6.09 6.75 6.01 7.82 5.96 6.60 6.64

Conductivity (mS/cm) ± ± ± ± ± ± ± ±

0.20 0.20 0.30 0.40 0.31a,b 0.50 0.62 0.62

± ± ± ± ± ± ± ±

16.02 18.05 17.99 18.13 18.43 17.93 18.68 19.35

0.59 0.35a 0.43a 0.39a 0.36a 0.43a 0.53a 0.90a

Density (g/mL) 1.17 1.10 1.08 1.03 1.11 1.03 1.05 1.02

± ± ± ± ± ± ± ±

0.02 0.07 0.04 0.06 0.04 0.06 0.08 0.06

Values are expressed as mean ± S.E.M. of five rats in each group in comparison with the control (a p < 0.05) or Tropaeolum majus extracts (HETM, TMLR) and ISQ (b p < 0.05) using one-way ANOVA followed by Bonferroni test.

B

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ACE inhibitory activity (% control)

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obtained in vivo, it appears to be related to inhibition of the angiotensin converter enzyme (ACE), and subsequent increase in the bioavailability of bradykinin, PGI2 and nitric oxide. In addition, an inhibitory effect on Na+ /K+ -ATPase was found in in vitro experiments and may also contribute to the increased diuresis induced by HETM, TMLR, and isoquercitrin from Tropaeolum majus.

C

administered in rats. This diuretic action was improved when a semi-purified fraction (TMLR) was used, and we found that it could be fully reproduced by administration of the flavonol isoquercitrin, a major component of this fraction (Gasparotto Junior et al., 2011a). In this study we investigated the mechanisms involved in the diuretic action of Tropaeolum majus and, accordingly our results

Fig. 2. Inhibitory effects of the Tropaeolum majus extracts (HETM and TMLR) and isoquercitrin (ISQ) on ACE activity (A) and serum aldosterone (B). The serum vasopressin values were not altered (C). All measurements were performed in samples from SHR rats after 7 days of treatment. Each bar represents the mean of five animals, and the vertical lines show the S.E.M. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Bonferroni test) (*p < 0.05; **p < 0.01; ***p < 0.001).

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B

A 100

Na+/K+/ATPase activity (% of control)

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100

100

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3 10 30 _ _________ _______ ____ HETM (μM)

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ISQ (μM)

TMLR (μM)

Fig. 3. Renal Na+ /K+ /ATPase inhibition after pre-incubation with HETM (A), TMLR (B), and ISQ (C). Each bar represents the mean ± S.E.M. of five experiments performed in triplicate. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Bonferroni test) (*p < 0.05; **p < 0.01).

B

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Fig. 4. Acute administration of HOE-140 reduces the diuretic response of HETM, TMLR and ISQ after 7 days of treatment. The graphs show the urinary volume (A) and sodium concentration (B) in samples collected for 15 h. Each bar represents the mean ± S.E.M. of five SHR rats. Asterisk denotes the significance levels of vehicle or HOE-140 in comparison with respective controls groups (one-way ANOVA followed by Bonferroni test). # denotes the significance levels compared with the groups that received HOE-140 (Student’s t-test) (# or *p < 0.05).

The effects of the main diuretic drugs result from direct inhibition of renal transporters, mainly located in the thick ascending limb of the loop of Henle (furosemide), and in the distal convoluted tubule (hydrochlorothiazide). Other diuretic drugs act inhibiting renal carbonic anhydrase (acetazolamide), blocking aldosterone receptors (spironolactone), or inhibiting epithelial Na+ channels

(amiloride). Moreover, ACE inhibitors such as captopril, indirectly increase diuresis due to reduction of aldosterone release, which is physiologically modulated by angiotensin II (Jackson, 1996). Despite the number of targets that can function for diuretic purposes, medicinal plants may be diuretic agents due to osmotic effects generated by large amounts of potassium and/or other

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Fig. 5. Acute administration of indomethacin abolishes the diuretic response of HETM, TMLR and ISQ after 7 days of treatment. Urinary volume (A) and sodium concentration (B) in samples collected for 15 h. Each bar represents mean ± S.E.M. of five SHR rats. Asterisk denotes the significance levels of vehicle or Indomethacin in comparison with respective controls groups (one-way ANOVA followed by Bonferroni test). # denotes the significance levels compared with the groups that received Indomethacin (Student’s t-test) (# or *p < 0.05).

A. Gasparotto Junior et al. / Journal of Ethnopharmacology 141 (2012) 501–509

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Fig. 6. Effects of acute administration of l-NAME on urinary volume (A) and sodium concentration (B) after 7 days of treatment with HETM, TMLR and ISQ. Each bar represents the mean ± S.E.M. of five SHR rats. Asterisk denotes the significance levels of vehicle or l-NAME in comparison with respective controls groups (one-way ANOVA followed by Bonferroni test). # denotes the significance levels compared with the groups that received l-NAME (Student’s t-test) (# or *p < 0.05).

metals found in these species (for mechanistic details see Loew, 1991). However, the quantitative determination of ions in the leaves of Tropaeolum majus revealed very low amounts of potassium and/or other metals, leading us to discard the possibility that its diuretic effect could be related to an osmotic mechanism. In addition, we observed that HETM, TMLR, and isoquercitrin, work as potassium-sparing agents, similarly to antagonists of aldosterone or epithelial Na+ -channel blockers. Taking into account the underlying mechanisms involved in the diuretic effects of clinically useful drugs, the first finding of this study was that the treatment with HETM, TMLR or isoquercitrin daily for 7 days did not change plasmatic vasopressin levels, but significantly inhibited the activity of ACE, leading to reduced amounts

of aldosterone in plasma. Although we had previously demonstrated that inhibition of ACE accounts for the hypotensive effects of HETM, TMLR and isoquercitrin after its acute administration, in here we provide evidence that inhibition of this protease is maintained even after continuous treatment for 7 days, indicating that no compensatory mechanisms or pharmacokinetic events were able to counteract this effect. The reduction in serum aldosterone by preparations obtained from Tropaeolum majus showed in this study, associated with the hypotensive action previously demonstrated (Gasparotto Junior et al., 2011b), may increase hydrostatic pressure in renal arterioles (due to vasodilation) and, together, be responsible for the diuretic and natriuretic effects observed in our study (Sakamoto et al., 1994; Gallieni et al., 2010).

Fig. 7. Isoquercitrin (ISQ) increases serum nitrite + nitrate (A) and reduces reactive oxygen species (ROS) in mesenteric artery of SHR rats (B). The serum and mesenteric arteries samples were obtained after 7 days of treatment. Each bar represents the mean ± S.E.M. of five animals. Asterisks denote the significance levels in comparison with the control group (one-way ANOVA followed by Bonferroni test) (*p < 0.05; **p < 0.01).

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ACE is also involved in kinin metabolism and the increased bioavailability of bradykinin has been often included in the beneficial clinical effects of ACE inhibitors (for review see Manolis et al., 2010). At least part of the natriuretic activity of ramipril (an ACE inhibitor used in humans) is generated by reduction of degradation of bradykinin (Sakamoto et al., 1994). Since the diuretic and natriuretic effects induced by HETM, TMLR or ISQ were abolished after administration of HOE-140, it is reasonable to suggest that the effects of this compound or extracts are mainly a consequence of increased bioavailability of bradykinin, as consequence of ACE inhibition. Accordingly to the vascular bed, activation of B2 bradykinin receptors expressed in endothelial cells may increase local production of PGI2 and NO, reducing vascular tone and blood pressure (Heitsch, 2003). In kidneys, locally produced prostaglandins are crucial mediators involved in maintainance of medullary hypertonicity, and regulation of blood perfusion and glomerular filtration rate (Kim et al., 2009). Drugs that release renal prostaglandins lead to increased renal blood flow, glomerular filtration rate, and renal sodium excretion (Stichtenoth and Frolich, 2000). Interestingly, pretreatment with the cyclooxygenase inhibitor indomethacin did not reduce the urinary volume nor Na+ excretion in untreated (control) rats, but avoided the diuretic and natriuretic effects of HETM, TMLR or ISQ. Stimulation of bradykinin B2 receptors in endothelial cells is also associated with release of NO, which has a central role in the maintenance of vascular tone and regulation of systemic blood pressure, exerting direct and indirect effects on kidney function. Independently of receptor activation, changes in oxidative stress may positively or negatively impair the bioavailability of NO, being determinant events in cardiovascular diseases (for review see Hirata et al., 2010). Recently, ACE inhibitors have been associated with reduction of oxidative stress and increased bioavailability of NO (Desideri et al., 2008; de la Rosa et al., 2010; Xia and Lazartigues, 2010). In addition, substantial evidence show the ability of polyphenolic compounds, including flavonoids such as quercetin, to inhibit ACE and simultaneously increase the release, or prevent degradation of NO (Ghosh and Scheepens, 2009; Perez-Vizcaino et al., 2009). Corroborating with these findings, we observed a significant increase in serum nitrite and nitrate in blood samples collected from spontaneously hypertensive rats treated with ISQ for 7 days. In addition, the treatment with the NO synthase inhibitor l-NAME avoided the diuretic effects of HETM, TMLR and ISQ. Moreover, mesenteric arteries from these same animals presented a marked reduction in ROS production, allowing us to suggest that an antioxidant effect of ISQ may increase the bioavailability of NO systemically (thus including kidneys), contributing to the diuretic effects of HETM, TMLR and ISQ. However, the relationship between these findings and the inhibition of ACE remains to be investigated. Another interestingly effect generated by ISQ and Tropaeolum majus extracts was the reduction in activity of renal Na+ /K+ -ATPase. In kidneys, inhibition of renal Na+ /K+ -ATPase can reduce the functionality of ionic carriers located in renal tubules, resulting in increased diuresis (Jackson, 1996; Gallieni et al., 2010). Although our data indicate a direct effect on renal Na+ /K+ -ATPase (as showed for other flavonols; e.g. Hirano et al., 1989; Zhu et al., 1997; Umarova et al., 1998), we cannot exclude that this inhibition may be related to ACE inhibition, as well as to increased NO bioavailability. Although several diuretics were available for use in humans, such drugs are associated with several adverse effects, including electrolyte and metabolic disorders (Ernst and Moser, 2009), reinforcing the importance of search for new molecules with diuretic properties and less adverse effects. Therefore, in recent years new diuretic drugs have been proposed mainly from natural products. Among these, the polyphenolic compounds have received special attention (Wright et al., 2007).

Isoquercitrin is not the first polyphenolic compound that is described as able to increase diuresis (Wright et al., 2007; Benjumea et al., 2009). However, none of these other compounds or phytoterapic medicines containing polyphenols had their diuretic effects linked to pathways involved in the effects of ISQ. Importantly, as well as in this study, oral administration of up to 5 g/kg of an aqueous infusion or an ethanolic extract obtained from the leaves of Tropaeolum majus did not show signs of acute toxicity (Zanetti et al., 2003; Gasparotto Junior et al., 2009). In summary, our results suggest that the mechanisms through ISQ (the main compound found in Tropaeolum majus extracts) exerts its diuretic effect in spontaneously hypertensive rats are mainly related to ACE inhibition, increased bioavailability of bradykinin, PGI2 and nitric oxide, which may involve an inhibitory effect on Na+ /K+ -ATPase. Despite the potent diuresis observed, isoquercitrin, as well as HETM and TMLR, act as potassium-sparing agents and their effects did not disappear even after 7 days of administration. Taken together, these findings support the potential of a preparation derived from Tropaeolum majus, or ISQ as a candidate to be a phytomedicine used in those cardiovascular diseases where increased diuresis is desired. Acknowledgements We are grateful to Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES-Brazil) and Diretoria Executiva de Gestão da Pesquisa e Pós-Graduac¸ão (DEGPP/UNIPAR-Brazil) for financial support. We also thank Dr. Douglas Cardoso Dragunski for Atomic Absorption Spectrometry (AAS) analysis. References Al-Dujaili, E.A., Mullins, L.J., Bailey, M.A., Kenyon, C.J., 2009. Development of a highly sensitive ELISA for aldosterone in mouse urine: validation in physiological and pathophysiological states of aldosterone excess and depletion. Steroids 74, 456–462. Benjumea, D., Martin-Herrera, D., Abdala, S., Gutierrez-Luis, J., Quinones, W., Cardona, D., Torres, F., Echeverri, F., 2009. Withanolides from Whitania aristata and their diuretic activity. Journal of Ethnopharmacology 123, 351–355. Brasil, 1998. Limites máximos de tolerância para contaminantes inorgânicos em alimentos Portaria ANVISA N◦ 685, de 27 de agosto de 1998, vol. 1, pp. 1–5. de la Rosa, A.P., Montoya, A.B., Martinez-Cuevas, P., Hernandez-Ledesma, B., LeonGalvan, M.F., De Leon-Rodriguez, A., Gonzalez, C., 2010. Tryptic amaranth glutelin digests induce endothelial nitric oxide production through inhibition of ACE: antihypertensive role of amaranth peptides. Nitric Oxide 23, 106–111. Desideri, G., Grassi, D., Croce, G., Bocale, R., Tiberti, S., Evangelista, S., Necozione, S., Di Orio, F., Ferri, C., 2008. Different effects of angiotensin converting enzyme inhibitors on endothelin-1 and nitric oxide balance in human vascular endothelial cells: evidence of an oxidant-sensitive pathway. Mediators of Inflammation, 305087. Ernst, M.E., Moser, M., 2009. Use of diuretics in patients with hypertension. The New England Journal of Medicine 361, 2153–2164. Fraige, K., Crespilho, F.N., Rezende, M.O.O., 2007. Determinac¸ão de zinco em solo utilizando colorimetria. Quimica nova 30, 588–591. Gallieni, M., Olivi, L., Mezzina, N., Cozzolino, M., Cusi, D., 2010. Renal effects of combined anti-hypertensive treatments. Recenti Progressi in Medicina 101, 70–77. Gasparotto Junior, A., Boffo, M.A., Lourenco, E.L., Stefanello, M.E., Kassuya, C.A., Marques, M.C., 2009. Natriuretic and diuretic effects of Tropaeolum majus (Tropaeolaceae) in rats. Journal of Ethnopharmacology 122, 517–522. Gasparotto Junior, A., Gasparotto, F.M., Boffo, M.A., Lourenco, E.L., Stefanello, M.E., Salvador, M.J., da Silva-Santos, J.E., Marques, M.C., Kassuya, C.A., 2011a. Diuretic and potassium-sparing effect of isoquercitrin—an active flavonoid of Tropaeolum majus L. Journal of Ethnopharmacology 134, 210–215. Gasparotto Junior, A., Gasparotto, F.M., Lourenco, E.L., Crestani, S., Stefanello, M.E., Salvador, M.J., da Silva-Santos, J.E., Marques, M.C., Kassuya, C.A., 2011b. Antihypertensive effects of isoquercitrin and extracts from Tropaeolum majus L.: evidence for the inhibition of angiotensin converting enzyme. Journal of Ethnopharmacology 134, 363–372. Ghosh, D., Scheepens, A., 2009. Vascular action of polyphenols. Molecular Nutrition & Food Research 53, 322–331. Heitsch, H., 2003. The therapeutic potential of bradykinin B2 receptor agonists in the treatment of cardiovascular disease. Expert Opinion on Investigational Drugs 12, 759–770. Hirano, T., Oka, K., Akiba, M., 1989. Effects of synthetic and naturally occurring flavonoids on Na+ ,K+ -ATPase: aspects of the structure–activity relationship and action mechanism. Life Sciences 45, 1111–1117.

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