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Involvement of bradykinin [B.sub.2] and muscarinic receptors in the prolonged diuretic and antihypertensive properties of Echinodorus grandiflorus (Cham. & Schltdl.) Micheli.


Background: Although Echinodorus grandiflorus (Cham. & Schltr.) Michel are used in Brazilian folk medicine as a diuretic drug, to date, no study has evaluated the mechanisms involved in this activity after prolonged administration in rats.

Aim of the study: Evaluate the possible mechanisms involved in the prolonged diuretic activity of ethanol soluble fraction obtained from Echinodorus grandiflorus (ES-EG) and to assess its relationship with hypotensive and antihypertensive activity using normotensive rats and those with renovascular hypertension (2K1C).

Methods: The diuretic effects of ES-EG (30-300 mg/kg; p.o.) were compared with hydrochlorothiazide in a repeated-dose treatment for 7 days. The urinary volume and sodium, potassium, chloride, bicarbonate contents, conductivity, pH and density were estimated in sample collected in 24 h for 7 days. Plasma sodium, potassium, total protein, urea, creatinine, aldosterone, vasopressin, nitrite, acetylcholinesterase concentration and angiotensin converting enzyme (ACE) activity were measured in samples collected at the end of the experimental period (seventh day). Using pharmacological antagonists or inhibitors, the involvement of bradykinin, prostaglandin, acetylcholine and nitric oxide (NO) in ES-EG-induced diuresis was determined. In addition, activities of erythrocytary carbonic anhydrase and renal [Na.sup.+]/K+/ATPase were evaluated in vitro.

Results: ES-EG increased diuresis similarly to hydrochlorothiazide and also presented HC[O.sub.3]-sparing effects and increased serum nitrite levels. Moreover, the intraduodenal administration of ES-EG induces significant hypotensive and antihypertensive effects in 2K1C rats. Previous treatment with HOE-140, indometacin and atropine fully avoided the diuretic effect of ES-EG, and including L-NAME pre-administration, it prevented the hypotensive and hypertensive activity induced by ES-EG. In addition, the association between HOE-140 and atropine or indometacin and L-NAME fully inhibited the hypotensive and antihypertensive effects of ESEG. The 7-day treatment with ES-EG resulted in increased plasma nitrite levels. All other parameters were not affected by treatment with ES-EG.

Conclusions: Our results suggest that the mechanisms through which Echinodorus grandiflorus extracts induce prolonged diuresis and reduce blood pressure in normotensive and 2K1C rats are mainly related to activation of muscarinic and bradykinin receptors with direct effects on prostaglandins and nitric oxide pathways.


Echinodorus grandiflorus





Nitric oxide


In recent decades, several studies have been conducted worldwide in order to evaluate the possible diuretic properties of different natural products. Most of these studies were only qualitative and not aimed at investigating the molecular mechanisms involved in these effects (Wright et al. 2007). Only in recent years, data emphasizing the mode of action of some diuretic plants and the relationship of these effects to their secondary metabolites have been published (de Souza et al. 2013; Gasparotto Junior et al. 2009, 2012; Leme et al. 2013).

In Brazil, several medicinal species are used as diuretic drugs, but most of them lack pharmacological studies showing the molecular pathways that might be contributing to these effects. Among them, Echinodorus grandiflorus (Cham. & Schltdl.) Micheli, a native species that occurs from southern Mexico to Brazil stands out. This plant belongs to family Alismataceae and sporadically grows as a plant cover on flooded or wet soils. In Brazil, it is popularly known as "chapeu de couro", "cha-de-campanha" or "erva do brejo", and its leaves are habitually used as a hot water infusion orally administered as diuretic, hypotensive, hypolipidemic, anti-inflammatory and analgesic (Bolson et al. 2015; Brugiolo et al. 2009; Lorenzi and Matos 2008). Recently, this species had its monograph described in the 5th edition of the Brazilian Pharmacopoeia (Brazil, 2011,2010) and the infusion obtained from its leaves has been indicated as mild diuretic and anti-inflammatory according to herbal form of the Brazilian Pharmacopoeia in its 1st edition (Brazil 2011).

Previous pharmacological studies have shown that extracts obtained from Echinodorus grandiflorus leaves induce antiedematogenic (Garcia et al. 2010), antihypertensive (Lessa et al. 2008), vasodilator (Tibiriga et al. 2007) and immunosuppressive (Pinto et al. 2007) effects, explaining, at least in part, its popular use against cardiovascular diseases. Previous phytochemical studies conducted with Echinodorus grandiflorus leaves have demonstrated the presence of fatty acids, diterpenoids, phenolic acids, flavonols, alkaloids, saponins and tannins, and their polyphenolic compounds have great potential to be the secondary metabolites responsible for their therapeutic properties (Brugiolo et al. 2009; Garcia et al. 2010).

Taking into account the popular use and previous evidence of effectiveness and therapeutic potential of Echinodorus grandiflorus against cardiovascular diseases, this study was conducted in order to investigate the molecular mechanisms involved in the possible diuretic effects induced by the aqueous extract obtained from Echinodorus grandiflorus and verify its relationship with a potential hypotensive and anti-hypertensive effect using normotensive rats and with renovascular hypertension (2K1C).

Material and methods


Hydrochlorothiazide (HCTZ; thiazide diuretic), acetazolamide (ACTZ; carbonic anhydrase inhibitor), ouabain octahydrate (sodium potassium ATPase inhibitor), N-[omega]-Nitro-L-arginine methyl ester (L-NAME; NO synthase inhibitor), icatibant acetate (HOE-140; [B.sub.2] bradykinin receptor antagonist), indometacin (cyclooxygenase inhibitor), atropine (muscarinic receptor antagonist), and captopril (angiotensin converting enzyme inhibitor), were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other drugs and reagents used were purchased from Merck (Darmstadt, Germany).

Plant material and preparation of aqueous Echinodorus grandiflorus extract

Echinodorus grandiflorus leaves were collected on February 2014 from the botanical garden of the Universidade Paranaense (UNIPAR) (Umuarama, Brazil) at 430 m altitude above sea level (S23[degrees]47'55-W53[degrees]18'48). A voucher specimen of this species is cataloged in the Herbarium of the Universidade Estadual de Maringa (HUEM) under number 20810.

The plant material was air-dried in an oven at 37 [degrees]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 was treated with 3 volumes of EtOH, which gave rise to a precipitate and an ethanol soluble fraction (ES-EG; yield 9.54%). All preparations were freeze-dried and maintained at room temperature until analyses.

Phytochemical investigation

The ethanol-soluble fraction (ES-EG) was analyzed by liquid chromatography-ultraviolet-mass spectrometry (LC-UV-MS) using an Acquity-UPLC (Waters) with LTQ-Orbitrap-XL (Thermo-Scientific). The chromatographic separation was developed on a C18 HSS T3 column (Waters) with 100 x 2.1 mm and 1.7 m of particle, at 60 [degrees]C. The solvent was composed of 0.1% formic acid (v/v) in water (A) and acetonitrile (B), with a gradient increasing solvent B: 0-30% in 13 min, -80% in 20 min, then returning to initial condition in 21 min, with flow rate of 400 [micro]l/min. The sample was prepared at 1 mg/ml and held at room temperature (22 [degrees]C), the injection volume was 2 [micro]l.

Detection was developed by photodiode-array at 200-400 nm and by mass spectrometry in positive and negative polarities, with mass range of 100-2000 m/z. The MS set up was: positive ions--spray voltage at 4.5 kV, capillary at 30 V, tube lens at 120 V, source temperature at 350 [degrees]C with nitrogen for desolvation at flow rate of 60 arbitrary units (a.u.) in sheath gas and 20 a.u. in auxiliary gas. For the negative ions, only voltages were different, being 3.5 kV on spray voltage, 10 V on capillary and 130 V on tube lens. For the structural characterization, fragmentation analyses were held by collision-induced dissociation (CID), with energy range of 20-25 eV. Mass accuracy was obtained by external calibration and the ion resolution was set at 7500 FWHM (full width at half maximum).

ES-EG sample was also analyzed by gas chromatography-mass spectrometry (GC-MS-Varian 4000) in order to characterize the composition of monosaccharides from glycosides observed by LC-UV-MS. ES-EG (1 mg) was previously hydrolyzed in 1 M trifluoroacetic acid for 12 h at 100 [degrees]C. The sample was dried at [N.sub.2] stream and the released monosaccharides were reduced with NaB[H.sub.4] to give their respective alditols, which were acetylated with 200 [micro]l of acetic anhydride and pyridine (1:1, v/v), for 12 h at room temperature. The reaction was stopped by addition of 200 [micro]l of methanol and the solvents were evaporated under [N.sub.2] stream. The resulting alditol acetates were analyzed by GC-MS with capillary column DB-225MS (30 m x 0.25 mm). The injector was held at 250 [degrees]C and column-oven heated from 50 to 220 [degrees]C, at 40 [degrees]C/min, for 20 min. Monosaccharide identification was based on the mass spectra fragmentation profile from electron ionization (70 eV), with ion trap analyzer, compared with authentic standards.

Pharmacological studies


Male Wistar rats (250-300 g) were used in experiments. The animals were provided by Universidade Federal do Parana (UFPR, Curitiba/PR, Brazil), and were kept in a temperature- and light-controlled room (22 [+ or -] 2 [degrees]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 Research Committee of Universidade Paranaense (UNIPAR, Brazil; authorization number 25454/2014).

Studies on renal function

Prolonged diuretic activity

The diuretic activity was determined accordingly to previous descriptions (Kau et al. 1984) with minor modifications. 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 (n = 5) were orally treated with ESEG (30,100 and 300 mg/kg) or hydrochlorothiazide (HCTZ; 25 mg/kg) by oral route (gavage) for 7 days. 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 every 24 h. Cumulative urine excretion was calculated in relation to body weight and expressed as ml/100 g. Cumulative electrolyte excretion load ([Na.sup.+], [K.sup.+], [Cl-.sup.-] and HC[O.sub.3.sup.-]), pH, density, and conductivity were estimated from urine samples from each rat. Plasma sodium, potassium, total protein, urea, and creatinine concentrations were also measured at the end of the experiment (7th day).

Analytical procedures

For serum analysis, blood samples were collected in conical tubes after decapitation. Plasma and serum were obtained by centrifugation (2000 rpm, 10 mins, 4 [degrees]C), and stored at -20 [degrees]C until analysis. Urinary and plasma [Na.sup.+] and [K.sup.+] levels were quantified by flame photometry (Quimis model Q398112). [Cl.sup.-] and HC[O.sub.3.sup.-] 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, Brazil), respectively. Density was estimated by weighing with Mettler AE163 ([+ or -] 0.1 mg) analytical scales on urine volume measured with Nichiryo micropipette. Plasma total protein, urea and creatinine levels were determined by enzymatic method for automated analyzer BM/Hitachi 912 (Cobas Mira, Roche, Indianapolis, USA). Additionally, excretion load (El) of sodium, potassium, chloride and bicarbonate was measured according to equation below (1):

EI = [U.sub.x]xV (1)

[U.sub.x], electrolytes concentration (mEq/1)

V, urinary flow (ml/min)

Results were expressed as [micro]Eq/min/100 g (1)

Evaluation of the mechanisms involved in the diuretic activity

Angiotensin converting enzyme (ACE) assay and determination of aldosterone, vasopressin and plasma cholinesterase activity

Samples were obtained from male rats after 7 days of treatment with ES-EG (30, 100 and 300 mg/kg), HCTZ (25 mg/kg) or vehicle (control). Blood was collected into glass tubes after decapitation and serum was obtained by centrifugation (800 g, 10 min, 4 [degrees]C). Aldosterone and vasopressin levels were measured by Enzyme linked immunosorbent assay (ELISA, Immuno-Biological Laboratories, Inc) and radioimmunoassay, respectively. Plasma cholinesterase activity was measured by automated colorimetric method (Cobas Mira, Roche, Indianapolis, USA).

ACE activity was determined as previously described (Santos et al. 1985). Serum (10 [micro]l was incubated for 15 min at 37 [degrees]C with 490 [micro]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 M). The production of His-Leu was fluorometrically measured (365 nm excitation and 495 emission, Aminco Model J4-7461 fluoromonitor, American Instrument Co., Silver Springs, MD) after the addition of 100 [micro]l of o-phthaldialdehyde (20 mg/ml in methanol), and 200 [micro]l of HCl (3 M), followed by centrifugation (800 g, 5 min) at room temperature. To correct intrinsic plasma fluorescence, zero-time blank samples were prepared by adding plasma after NaOH treatment. In some experiments, captopril (60 mg/kg; orally for seven days) was used as positive control. All measurements were performed in triplicate.

Measurement of the erythrocyte carbonic anhydrase activity

After the 7 days of treatment with vehicle, ES-EG (30, 100 and 300 mg/kg), or acetazolamide (25 mg/kg), blood samples were collected in tubes containing EDTA sodium. The red blood cells (RBC) were obtained by centrifugation at 1500-2000 x g for 10-15 min at room temperature. One ml of RBC 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 at ratio of 1:10 (v/v) in distilled water, resulting in hemolysate at dilution of 1/40 (v/v). Two hemolysate samples (50 [micro]l) were incubated with alpha-naphthyl 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 [degrees]C for 20 min. The second sample was exposed to acetazolamide at the end of the incubation period (37 [degrees]C for 20 min). Then, 500 [micro]l of 5-chloro-o-toluidine (2 mg/ml) was added to the samples and, after 15 mins, the absorbance was measured at 555 nm. Each 1 mmol of acetate alphanaphthol produced per minute per ml of hemolysate was considered as one unit (U) (Tashian 1989).

Renal [Na.sup.+]/[K.sup.+]/ATPase activity

The [Na.sup.+]/[K.sup.+]/ATPase activity was determined by the Noel and Godfraind (1984) method, using kidney samples obtained from normotensive rats. The specific enzyme activity corresponds to the difference between the total ATPase activity and the activity measured in the presence of 1 mM ouabain (ouabain-resistant activity). The amount of protein was adjusted in order to hydrolyze no more than 10-15% of substrate during the incubation period. The reaction was started by addition of kidney samples, incubated at 37 [degrees]C for 2 h, in a total volume of 0.5 ml. Incubation was performed in the presence of 84 mM NaCl, 3 mM KCl, 3 mM Mg[Cl.sub.2], 1.2 mM ATP[Na.sub.2], 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 ES-EG concentrations (3-30 [micro]M). The extract concentration was estimated in experiments previously conducted (Gasparotto Junior et al. 2012).

The role of the nitric oxide (NO), acetylcholine, bradykinin, and prostaglandins system in the diuretic effects of ES obtained from Echinodorus grandiflorus

This study was performed accordingly to previous descriptions (Gasparotto et al. 2009, 2012). After receiving oral administration of isotonic saline (0.9% NaCl, 5 ml/100 g) to impose a controlled water and salt load, different groups of rats received L-NAME (60 mg/kg, given 1 h before the experiments; p.o.), atropine (1 mg/kg, given 1 h before the experiments; p.o.), HOE-140 (1.5 mg/kg, given 15 min before; i.p.), or indometacin (5 mg/kg, given 1 h before; p.o,), followed by oral administration of deionized water (5 ml/kg, control group), ESEG (300 mg/kg), or hydrochlorothiazide (25 mg/kg). The total volume of urine output was measured 8 h after treatments. Sodium, potassium, chloride and bicarbonate concentrations, as well as pH, density and conductivity of urine samples were evaluated.

Determination of serum nitrate/nitrite (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 (8 h 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 [degrees]C for 2 h in the presence of nitrate reductase expressed in Escherichia coli. After the incubation period, samples were centrifuged (800 g, 10 mins) to remove bacteria. Then, 100 [micro]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 nitrite and nitrate curves (0-150 mM) were performed simultaneously.

Studies on systemic blood pressure

Induction of renovascular hypertension (two-kidney, one-clip model)

The Goldblatt 2K1C hypertension model was induced according to procedure described by Umar et al. (2010). Briefly, rats were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), intraperitoneally administered. The left renal artery was exposed by retroperitoneal flank incision and dissected free of the renal vein and connective tissue. A silver clip with 0.22 mm alumen was placed around the artery for partial occlusion; in sham operations, the artery was not clipped. After 6 weeks, systolic blood pressure (SBP) was measured using the tail-cuff method in conscious rats. Only hypertensive rats (2K1C; SBP above 150 mm Hg) were used in experiments.

Direct blood pressure measurement in anesthetized rats

Normotensive male rats were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg), intramuscularly administered and supplemented at 45-60 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. i.v.) was 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[R] recording system, and an application program (Chart, v 4.1; all from ADI Instruments; Castle Hill, Australia), which recorded the mean arterial pressure (MAP). For intraduodenal administration, a small incision was made below the xiphoid process and the duodenum was isolated with the aid of tweezers. For blood pressure stabilization after the surgical process, an interval of 15 mins was given before recordings. At the end of experiments, animals were euthanized with an overdose of thiopental (over 40 mg/kg, i.v.).

Hypotensive and antihypertensive dose response of ES-EG

Groups of normotensive and 2K1C rats, prepared for direct blood pressure measurements as previously described, received intraduodenal ES-EG at 30,100 and 300 mg/kg, respectively. The control group received intraduodenal vehicle at constant volume of 100 [micro]/100 g of body weight (BW). Changes in MAP were recorded for 45 min after treatments.

Participation of NO, acetylcholine, bradykinin, and prostaglandins in the hypotensive and antihypertensive response of ES-EG

In this set of experiments, animals were prepared for MAP recording as previously described. Different groups of rats received L-NAME (60 mg/kg, given 1 h before experiments; p.o.), atropine (1 mg/kg, given 1 h before experiments; p.o.), HOE-140 (1.5 mg/kg, given 15 min before; i.p.) or indometacin (5 mg/kg, given 1 h before; p.o.), followed by intraduodenal administration of ES-EG (300 mg/kg) or vehicle (100 [micro]l/100 g BW). The effect of association between HOE-140 and atropine or indometacin and L-NAME was also evaluated under the same experimental conditions previously described. Changes in MAP were recorded for 45 min after treatments.

Statistical analyses

Results were expressed as mean [+ or -] standard error mean (S.E.M.) of 5 animals per group. Statistical analyses were performed using one or two-way analysis of variance (ANOVA) followed by Bonferroni's or Student's t-test, when applicable. P-value less than 0.05 was considered statistically significant. Graphs were drawn and statistical analysis was carried out using GraphPad Prism version 5.0 for Mac OS X (GraphPad Software, San Diego, CA, USA).


Phytochemical characterization

The analysis was complex due to the presence of many compound/peaks on chromatogram (Fig. 1A). Using MS detection, many compounds were identified, including the presence of disaccharide at m/z 341.10873 [[M-H].sup.-], probably sucrose, commonly in this kind of extraction. Other minor components were consistent with feruloyltartaric acid (m/z 325.05678 - [lambda] 302, 327) and diferuloyl-tartaric acid (m/z 501.10386 - [lambda] 302, 327). Nevertheless, most compounds observed had characteristic UV-absorption at [lambda] ~270 and ~346, typical of some flavonoids, which were further identified by MS, being consistent with flavone-C-glycosides. Additional analysis was developed by GC-MS in order to obtain the monosaccharide composition, determined from their volatile alditol acetate derivatives. The composition was: rhamnose (5%), ribose (4%), arabinose (5%), xylose (3%), galactose (10%), glucose (32%), myo-inositol also appears at high amounts, 41%.

In contrast to flavonoid O-glycosides, C-glycosides have a different fragmentation profile, with intra-glycosidic ring cleavage instead of glycosidic linkage breakdown, making their analysis more difficult. Characteristically, negative fragments from C-hexosides appear with 2 neutral losses (NL), 90 and 120 mass units (m.u.), arising from [sup.0,2]X and [sup.0,3]X fragments (see Domon and Costello, 1988 for glycoside fragments nomenclature). An additional loss of [H.sub.2]O (NL 18.010 m.u.) was observed, and from methoxylated flavonoids, neutral losses of [CH.sub.3] (NL 15.024 m.u.), [C.sub.2][H.sub.4] (NL 28.031 m.u.) and [C.sub.2][H.sub.3]O (NL 43.019 m.u.) were also observed. Many compounds from ES-EG exhibited this characteristic fragmentation profile on negative ionization MS-analysis (Fig. 1B-G), being identified as (5) isoorientin (Rt 7.52,m/z 447.09334), (7) swertiajaponin (Rt 7.82, m/z 461.10887), (10) isovitexin (Rt 8.51, m/z 431.09840), (12) swertisin (Rt 8.86, m/z 445.11430) and (15) isoorientin 7,3'-dimethylether (Rt, 9.40, m/z 475.12557). This identification is in agreement with previous findings (Schnitzler et al. 2007).

Although these flavone C-mono-glucosides were reported in Echinodorus grandiflorus, several other compounds from ES-EG fraction had m/z values consistent with 2 glycosyl units linked to the flavonoid, and CID-MS analysis indicates that the second glycosyl seems to be O-linked (Fig. 8A-Supplementary material). These diglycosides appear at high abundance, comprising major compounds detected from ES-EG. Compound (6) provided ion at m/z 593.15135 with fragments at m/z 503.121 ([sup.0,3][X.sub.1]), 473.109 ([sup.0,2][X.sub.1]), 429.082 ([Z.sub.1]), 339.050 ([sup.0,3][X.sub.0]) and 309.039 ([sup.0,2][X.sub.0]). These fragments indicate loss of 90 and 120 m.u., typical from C-hexosides, but fragment m/z 429.08 is consistent with [Z.sub.1] type, produced by removal of a deoxyhexosyl unit (NL 164.068). Thus, compound (6) is consistent with isoorientin-O-rhamnoside (Fig. 8B--Supplementary material). Compound (8) gave ion at m/z 607.16698 and fragments of m/z 487.125 ([sup.0,2][X.sub.1]), 461.108 ([Y.sub.1]), 443.098([Z.sub.1]), 341.066 ([sup.0,2][X.sub.0]) and 323.055 ([sup.0,2][X.sub.0]). In this case, since fragments from Z and Y series were produced, 2 [sup.0,2]X ions were observed, with difference of 18.010 m.u., consistent with water, as observed from [Y.sub.1] and [Z.sub.1]. Compound (8) was identified as swertiajaponin-0-rhamnoside (Fig. 8C--Supplementary material).

The major peak from ES-EG chromatogram was compound (9), which gave the ion at m/z 577.15633, with fragments at m/z 457.114 ([sup.0,2][X.sub.1]), 413.088 ([Z.sub.1]), 323.055 ([sup.0,3][X.sub.0]) and 293.045 ([sup.0,2][X.sub.0]). [Y.sub.1] ion appears at low abundance at m/z 431.097. This fragmentation profile was consistent with isovitexin-O-rhamnoside (Fig. 8D-Supplementary material). Compound (11) with m/z 591.17193 and fragments at m/z 471.129 ([sup.0,2][X.sub.1]), 445.114 ([Y.sub.1]), 427.103 ([Z.sub.1]), 325.071 ([sup.0,2][X.sub.0]) and 307.061 ([sup.0,2][X.sub.0]) was consistent with swertisin-O-rhamnoside (Fig. 8E-Supplementary material). Again, the presence of two [sup.0,2][X.sub.0] ions is due to the formation of [Y.sub.1] and [Z.sub.1] fragments. Compound (13) is an isomer of compound (8), with m/z 607.16711, but its fragment was slightly different, at least in abundance. A small [sup.0,2][X.sub.1] fragment at m/z 487.123 was accompanied by the highest [Z.sub.1] ion at m/z 443.098. [Y.sub.1] ion was almost absent, thus only one [sup.0,2][X.sub.0] fragment at m/z 323.056 was observed, together with [sup.0,3][X.sub.0] ion at m/z 353.067. Thus, compound (13) is an isomer of swertiajaponin-O-rhamnoside (Fig. 8F--Supplementary material). Compound (14) produced ion at m/z 621.18263 and fragments at m/z 606.158 (M-C[H.sub.3]), 501.140 ([sup.0,2][X.sub.1]) 475.124 ([Y.sub.1]) 457.114 ([Z.sub.1]), 442.090 ([Z.sub.1]-C[H.sub.3]) and 337.072 ([sup.0,2][X.sub.0]). These fragments are consistent with isoorientin-O-rhamnoside 7,3'-dimethylether (Fig. 8 G-Supplementary material). Other compounds present on chromatogram could not be identified (Table 2--Supplementary material).

Prolonged diuretic activity

Effect on urinary volume

Daily administration of ES-EG (300 mg/kg) for 7 days significantly increased diuresis after the second day of treatment (Fig. 2), remaining until the seventh day. At this point, the cumulative urinary flow increased from 25.63 [+ or -] 1.09 ml in control animals to 35.72 [+ or -] 3.25 ml in rats treated with ES-EG. The cumulative urinary volume found in ES-EG-treated animals during all experimental period was not different than values obtained in animals treated with HCTZ, a classical diuretic agent.

Effect on urinary electrolyte excretion

The effects of prolonged treatment with ES-EG (30, 100 and 300 mg/kg) and hydrochlorothiazide on electrolyte ([Na.sup.+], [K.sup.+], [Cl.sup.-], and HC[O.sub.3.sup.-]) excretion in urine are presented in Figs. 3. Urinary excretion of sodium, potassium (from the first day) and chloride (after the second day) were significantly increased in groups treated with ES-EG (300 mg/kg) when compared to controls treated with vehicle alone (Fig. 3A-C). Similarly, urinary conductivity remained high in groups treated with HCTZ and ES-EG (300 mg/kg) throughout the treatment period.

Although urinary sodium, potassium and chloride excretion induced by ES-EG did not differ from values observed with the use of hydrochlorothiazide, group treated with only this classical diuretic showed increased amounts of HC[O.sub.3] in urine (Fig. 3D). Moreover, the smaller ES-EG doses (30 and 100 mg/kg) did not affect the urinary concentration of any electrolyte measured. All other evaluated parameters (pH and density) did not show significant differences when compared to the control group (data not shown).

Effect on urea, creatinine, total protein and electrolyte plasma levels

The plasma creatinine, urea, total protein, [Na.sup.+] and [K.sup.+] levels measured at the end of the experiment (7th day) were not affected by any of the drugs tested (data not shown).

Evaluation of mechanisms involved in the diuretic activity

ES-EG treatment does not affect serum aldosterone, vasopressin levels or ACE and cholinesterase activity

None of the groups treated with ES-EG (30-300 mg/kg) showed significant differences in the determination of serum vasopressin, aldosterone levels or plasma ACE and cholinesterase activity when compared to the control group. On the other hand, as expected, positive control captopril (60 mg/kg) reduced the plasma ACE activity and serum aldosterone levels by approximately 40% (Table 1).

Prolonged treatment with ES-EG did not affect erythrocyte carbonic anhydrase or renal [Na.sup.+]/[K.sup.+]/ATPase activity

Treatment with all tested drugs (ES-EG or HCTZ) did not change the erythrocyte carbonic anhydrase activity, while the administration of acetazolamide, a classic carbonic anhydrase inhibitor, reduced it by 46 [+ or -] 7%. Moreover, incubation of ES-EG (3-30 [micro]M) in kidney samples obtained from normotensive rats has not been able to change the in vitro activity of renal [Na.sup.+]/[K.sup.+]/ATPase (Fig. 9B-C--Supplementary material).

Bradykinin [B.sub.2] and muscarinic receptors blockade prevents diuresis, natriuresis, kaliuresis, and chloride excretion induced by ES-EC

Previous administration of HOE-140 fully inhibited diuretic, natriuretic and [K.sup.+] and [Cl.sup.-] excretion induced by ES-EG in normotensive rats (Fig. 4A-E). A similar inhibitory effect was found in animals previously treated with atropine (Fig. 4F-J). Moreover, all other parameters evaluated (pH, conductivity and density) did not show significant differences when compared to the control group (data not shown).

ES-EG induced diuresis and electrolyte excretion depends on the prostaglandin pathway

Under pharmacological blockade of prostaglandin synthase, ESEG-induced diuresis and electrolyte excretion were reduced to levels similar to those obtained with animals treated only with vehicle (Fig. 5A-E). However, under L-NAME administration, the ability of ES-EG to induce diuresis was not significantly inhibited (Fig. 5F-J).

Seven days of treatment with ES-EG increase serum nitrite in normotensive rats

Daily treatment with ES-EG (300 mg/kg) significantly increased serum nitrite levels when compared to animals treated only with vehicle (ES-EG: 90 [+ or -] 6.6 [micro]M; control: 59 [+ or -] 4.8 [micro]M) (Fig. 9A--Supplementary material).

Hypotensive and antihypertensive effects of ES-EG on normotensive and 2K1C hypertensive rats

The basal MAP recorded in normotensive and 2K1C hypertensive rats after the 15-min period allowed for stabilization and before the administration of any drug, was 100.9 [+ or -] 4.0 mm Hg and 140.5 [+ or -] 3.3 mm Hg, respectively. Intraduodenal administration of ESEG caused a dose dependent hypotension (Fig. 6A), which lasted for 2.6 [+ or -] 0.4 and 7.8 [+ or -] 0.6 mm Hg (100 and 300 mg/kg. respectively), with minor effects on heart rate (data not shown). On the other hand, in 2K1C hypertensive rats, the administration of ES-EG at doses of 100 and 300 mg/kg reduced MAP levels in 3.2 [+ or -] 0.6, and 11.0 [+ or -] 1.1 mm Hg, respectively (Fig. 6F).

Hypotensive and antihypertensive effects of ES-EG is mediated by activation of bradykinin [B.sub.2] and muscarinic receptors with direct participation of prostaglandin and NO pathway

ES-EG-induced hypotension in normotensive (Fig. 6B-E) and 2K1C hypertensive rats (Fig. 6G-J) was decreased by up to 50% under pharmacological blockade of bradykinin [B.sub.2] and muscarinic receptors or after direct inhibition of prostaglandin and nitric oxide synthesis. On the other hand, after association between [B.sub.2] and muscarinic receptors antagonists or prostaglandins and nitric oxide synthesis inhibitors, the hypotensive and antihypertensive effects were completely inhibited (Fig. 7A-D).


The interest in medicinal plants and their extracts grows every year, and therefore, several natural products become direct targets of research to develop new diuretic and antihypertensive drugs (Gutierrez and Baez 2009; Wright et al. 2007). In Brazil, the extensive popular use of Echinodorus grandiflorus leaves and their inclusion in official forms characterizes this species as promising for ethnopharmacological survey of their important biological activities commonly described (Bolson et al. 2015; Brazil 2011; Brugiolo et al. 2009; Lorenzi and Matos 2008). Data obtained in this study have demonstrated for the first time that a preparation obtained from the infusion of this species (ES-EG) may exhibit prolonged diuretic activity, and hypotensive and antihypertensive effects when administered by the enteral route, and show that these effects appear to be mediated by bradykinin [B.sub.2] and muscarinic receptors inducing the release of endothelial NO and prostaglandins.

In recent years, it has been described in literature that different extracts obtained from Echinodorus grandiflorus might cause antihypertensive and vasodilator effect through the release of endothelial NO with direct participation of muscarinic receptors (Lessa et al. 2008; Tibirica et al. 2007). So, when carried out this study, we assumed that some secondary metabolites present in ES-EG could be affecting or influencing the bioavailability of endogenous NO. At first, considering the massive presence of polyphenolic compounds in the ES-EG (mainly flavones), it was decided to investigate whether ES-EG would be able to induce in vivo antioxidant effects. Data revealed that the renal and cardiovascular effects of ES-EG could present an important contribution of a direct or indirect endogenous antioxidant effect, because the increased serum nitrite may indicate a significant increase in the bioavailability of endogenous NO.

Despite some evidence suggesting a reduction in the degradation of endogenous NO due to the antioxidant properties of ES-EG, it was also found that some endogenous mediator, such as bradykinin and acetylcholine, may be involved in release of endothelial NO. Classically, it is known that both bradykinin and acetylcholine through their respective [B.sub.2] and [M.sub.3] receptors may influence the synthesis and release of vasorelaxant, anti-hypertrophic and anti-atherosclerotic endothelial mediators. A significant increase in endothelial calcium stimulating eNOS and prostaglandin synthase occurs after activation of these receptors, which culminates with the release of several mediators, including NO, prostaglandins and tissue-type plasminogen activator.

NO and prostaglandins play an important role in the systemic and renal arterioles. While in arterial microcirculation, prostaglandins and especially NO play a key role in regulating arteriolar tone and blood pressure, in renal level, these mediators regulate the dilation of the afferent arterioles and glomerular filtration rate (Chappell 2012). Therefore, substances that directly or indirectly increase the release of these mediators can result in increased renal elimination of salt and water and induce an important systemic vasodilation and hypotension (Heitsch 2003; Katori and Majima 2014).

In this study, the fact that bradykinin [B.sub.2] and muscarinic receptor antagonists reduce the diuretic, hypotensive, and antihypertensive effects of ES-EG, suggests that these effects are also indirectly dependent on the release of endothelial NO and prostaglandins. In fact, the administration of indomethacin or L-NAME reduced substantially the cardiovascular and renal properties of ES-EG. More than that, the association between bradykinin [B.sub.2] and muscarinic receptors antagonists or inhibitors of the synthesis of NO and prostaglandins completely blocked the hypotensive and antihypertensive effects of ESEG, showing an interrelated effect among bradykinin, acetylcholine, NO and prostaglandins.

In recent years, several studies have reported that plants rich in polyphenols, particularly free flavonoids and their glycosylated derivatives, may induce diuretic, hypotensive, and antihypertensive effects through the release of endothelial NO and prostaglandins, mainly mediated by ACE inhibition and activation of bradykinin [B.sub.2] receptors (de Souza et al. 2013; Gasparotto Junior et al. 2012, 2009; Leme et al. 2013). Since study did not detect any sign of inhibition of metabolic breakdown of bradykinin by angiotensin-converting enzyme inhibition, we believe that the large presence of these polyphenols in ES-EG may have significantly contributed to the activation of bradykinin [B.sub.2] receptors with final release of NO and prostaglandins. On the other hand, the fact that ES-EG could stimulate additionally and simultaneously the cholinergic pathway in vascular and renal level drew our attention. Although we believe that the vascular effects of ES-EG may be due to the stimulation of [M.sub.3] receptors in the vascular endothelium, we do not exclude the possibility that other cholinergic receptors may also be involved, mainly because we did not detect any evidence of acetylcholinesterase inhibitory activity by the ES-EG.

In short, this study demonstrates that the ES-EG obtained from Echinodorus grandiflorus leaves can produce significant prolonged diuretic activity accompanied by hypotensive and antihypertensive effects. The mechanisms involved in this activity were critically investigated and suggest the direct participation of bradykinin [B.sub.2] and muscarinic receptors in the release of NO and prostaglandins. Moreover, oral treatment with the ES-EG induces effects similar to hydrochlorothiazide with the advantage of not inducing a significant loss of bicarbonate in the urine. Data presented highlight the effectiveness of ES-EG obtained from Echinodorus grandiflorus and show that this species is a potential candidate for the development of a new herbal medicine for the treatment of renal and cardiovascular diseases when diuretic and hypotensive effects are required.


Article history:

Received 19 August 2015

Revised 27 October 2015

Accepted 30 October 2015

Conflict of interest

The authors declare that there are no conflicts of interest in this study.


We are grateful to Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES-Brazil), Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq-Brazil), Fundacao de Apoio ao Desenvolvimento do Ensino, Ciencia e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT-Brazil), and Diretoria Executiva de Gestao da Pesquisa e Pos-Graduacao (DEGPP/UNIPAR-Brazil) for financial support.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2015.10.020.


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Thiago Buno Lima Prando (b), Lorena Neris Barboza (b), Valdinei de Oliveira Araujo (b), Francielly Mourao Gasparotto (a), Lauro Mera de Souza (c, 1), Emerson Luiz Botelho Louren, (b) Arquimedes Gasparotto Junior (a), *

(a) Laboratario de Farmacologia Cardiovascular, Faculdade de Ciencias da Saude, Universidade Federal da Grande Dourados, Rodovia Dourados-Itahum, km 12, P.O. Box 533, 79.804-970, Dourados, MS, Brazil

(b) Laboratario de Farmacologia e Toxicologia de Produtos Naturais, Universidade Paranaense, 4282-Centro, Umuarama-PR, 87502-210, Brazil

(c) Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Parana, P.O. Box 19046, 81.531-980, Curitiba, PR, Brazil

Abbreviations: ACE:, angiotensin-converting enzyme; ACTZ:, acetazolamide; ANOVA:, analysis of variance; BW:, body weight; [degrees]C:, degree Celsius; CID:, collision-induced dissociation; CG-MS:, chromatography-mass spectrometry; EDTA:, ethylene-diaminetetraacetic acid; El:, excretion load; ELISA:, enzyme linked immunosorbent assay; eV:, electron-volts; ES-EG:, ethanol soluble fraction obtained from Echinodorus grandiflorus: ETOH:, ethanol; FWHM:, full width at half maximum; HOE-140:, icatibant acetate; HR:, heart rate; HCTZ:, hydrochlorothiazide; HUEM:, herbarium of the Universidade Estadual de Maringa; INDO:, indometacin; LC-UV-MS:,

chromatography-ultra-violet-mass spectrometry, L-NAME:, N-[omega]-nitro-L-arginine methyl ester; MAP:, mean arterial pressure; NO:, nitric oxide; NOx:, nitrate/nitrite serum; [PGI.sub.2]:, prostacyclin; RBC:, red blood cells; ROS:, reactive oxygen species; SBP:, systolic blood pressure; SEM:, standard error of the mean; UFPR:, Federal University of Parani; UNIPAR:, Universidade Paranaense; UPLC:, ultra performance liquid chromatography; Ux:, electrolytes concentration; V:, volts; 2K1C:, two-kidney, one-clip Goldblatt hypertensive rats.

* Corresponding author. Tel.: +55 67 3410 2333; fax: +55 67 3410 2321.

E-mail addresses: (L.M. de Souza),, (A. Gasparotto Junior).

(1) Tel.: +55 41 3361 1577; fax: +55 41 3266 2042.

Table 1
Effect of prolonged oral administration of ES obtained from
Echinodorus grandiflorus on serum vasopressin, aldosterone and plasma
ACE and cholinesterase activity.

Group                  Plasma ACE activity   Serum cholinesterase
                       (mmol/[min.sup.-1]/   activity (IU/1)

Control                158 [+ or -] 8.5      8439 [+ or -] 654
HCTZ(25 mg/kg)         161 [+ or -] 7.0      7925 [+ or -] 764
Captopril (60 mg/kg)   95 [+ or -] 9.0 (a)   8661 [+ or -] 749
ES-EG (30 mg/kg)       164 [+ or -] 8.2      9123 [+ or -] 824
ES-EG (100 mg/kg)      150 [+ or -] 7.9      7564 [+ or -] 788
ES-EG (300 mg/kg)      153 [+ or -] 9.1      8945 [+ or -] 899

Group                  Vasopressin         Aldosterone
                       (pmol/1)            (pg/ml)

Control                6.4 [+ or -] 1.74   142 [+ or -] 4.8
HCTZ(25 mg/kg)         6.3 [+ or -] 1.58   147 [+ or -] 6.3
Captopril (60 mg/kg)   6.4 [+ or -] 1.99   102 [+ or -] 6.3 (a)
ES-EG (30 mg/kg)       5.9 [+ or -] 1.88   141 [+ or -] 5.7
ES-EG (100 mg/kg)      6.1 [+ or -] 1.50   150 [+ or -] 4.3
ES-EG (300 mg/kg)      6.6 [+ or -] 2.00   149 [+ or -] 6.3

Values are expressed as mean [+ or -] S. E. M. of five rats in each
group in comparison with the control using one-way ANOVA followed by
Bonferroni's test ((a) p < 0.05).


Please note: Some tables or figures were omitted from this article.
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Author:Prando, Thiago Buno Lima; Barboza, Lorena Neris; Araujo, Valdinei de Oliveira; Gasparotto, Franciell
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 15, 2016
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