Research ArticleRoles of Nitric Oxide and Prostaglandins in the SustainedAntihypertensive Effects of Acanthospermum hispidum DC onOvariectomized Rats with Renovascular Hypertension

Rhanany Alan Calloi Palozi1 Maysa Isernhagen Schaedler1

Cleide Adriane Signor Tirloni1 Aniely Oliveira Silva1

Francislaine Aparecida dos Reis Liacutevero2 Roosevelt Isaias Carvalho Souza1

Ariany Carvalho dos Santos1 Thiago Bruno Lima Prando2

Lauro Mera de Souza3 and Arquimedes Gasparotto Junior1

1Faculdade de Ciencias da Saude Universidade Federal da Grande Dourados Rodovia Dourados-ItahumKm 12 PO Box 533 79804970 Dourados MS Brazil2Laboratorio de Pesquisa Pre-Clınica de Produtos Naturais Universidade ParanaensePraca Mascarenhas de Moraes 4282 PO Box 224 87502-210 Umuarama PR Brazil3Instituto de Pesquisa Pele Pequeno Prıncipe Faculdades Pequeno Prıncipe Av Iguacu 333 80230020 Curitiba PR Brazil

Correspondence should be addressed to Arquimedes Gasparotto Junior arquimedesgasparottogmailcom

Received 28 June 2017 Revised 10 August 2017 Accepted 16 August 2017 Published 20 September 2017

Academic Editor Jian-Li Gao

Copyright copy 2017 Rhanany Alan Calloi Palozi et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Although Acanthospermum hispidum is used in Brazilian folk medicine as an antihypertensive no study evaluated itseffects on a renovascular hypertension and ovariectomy model So this study investigated the mechanisms involved in theantihypertensive effects of an ethanol-soluble fraction obtained fromA hispidum (ESAH) using two-kidney-one-clip hypertensionin ovariectomized rats (2K1C plus OVT) ESAH was orally administered at doses of 30 100 and 300mgkg daily for 28 days after5 weeks of surgery Enalapril (15mgkg) and hydrochlorothiazide (25mgkg) were used as standard drugs Diuretic activity wasevaluated on days 1 7 14 21 and 28 Systolic diastolic and mean blood pressure and heart rate were recorded Serum creatinineurea thiobarbituric acid reactive substances nitrosamine nitrite aldosterone vasopressin levels and ACE activity were measuredThe vascular reactivity and the role of nitric oxide (NO) and prostaglandins (PG) in the vasodilator response of ESAH on themesenteric vascular bed (MVB) were also investigated ESAH treatment induced an important saluretic and antihypertensiveresponse therefore recovering vascular reactivity in 2K1C plus OVT-rats This effect was associated with a reduction of oxidativeand nitrosative stress with a possible increase in the NO bioavailability Additionally a NO and PG-dependent vasodilator effectwas observed on the MEV

1 Introduction

In recent years evidence has shown that there are significantdifferences between the genesis and the development ofcardiovascular diseases between men and women [1] Ingeneral women are more affected by cardiovascular diseasesespecially hypertension after a pronounced drop in estrogenlevels a fact that usually occurs after menopause It is now

known that one of the mechanisms by which blood pressuremay be elevated in aging postmenopausal women is theactivation of the renin-angiotensin system (RAS) Post-menopausal women exhibit an increase in plasma reninactivity suggesting activation of the RAS similarly to whatoccurs during renovascular hypertension [2] Approximately50 of menopausal women have hypertension a conditionthat associated with other estrogen-related risk factors (such

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2492483 14 pageshttpsdoiorg10115520172492483

2 Evidence-Based Complementary and Alternative Medicine

as obesity and dyslipidemia) significantly contributes to acutecardiovascular events [3] In contrast although this conditionis quite common preclinical studies specifically designed forthis type of population are still very restricted [4]

Recently we have shown that the ethanol-soluble fractionobtained from Acanthospermum hispidum DC (Asteraceae)(ESAH) an important medicinal species widely used inBrazil has a significant acute hypotensive effect on normot-ensive male Wistar rats [5] In addition we have also shownthat ESAH rich in phenolic compounds such as derivativesof caffeic acid and glycosylated flavonoids (quercetin glu-coside and galactoside) does not produce any toxic effectsafter acute treatment [5] So despite the widespread useof A hispidum as an antihypertensive by the Brazilian popu-lation [6] its prolonged diuretic and antihypertensive effectshave not yet been scientifically evaluated

Thus in this work the prolonged diuretic and antihyper-tensive effects of A hispidum on ovariectomized rats withrenovascular hypertension were investigated to simulate abroad part of the woman population aged over 50 years Inaddition the molecular mechanisms involved in A hispidumantihypertensive response using isolated mesenteric vascularbed (MVB) were also evaluated

2 Materials and Methods

21 Drugs The following drugs salts and solutions wereused xylazine and ketamine hydrochloride (from SyntecSao Paulo SP Brazil) heparin (from Hipolabor Belo Hor-izonte MG Brazil) and acetylcholine chloride phenyle-phrine indomethacin N120596-nitro-L-arginine methyl ester(L-NAME) sodium nitroprusside NaCl KCl NaHCO3MgSO4 CaCl2 KH2PO4 dextrose and ethylenediaminete-traacetic acid (EDTA) (all purchased from Sigma-AldrichSaint Louis MO USA)

22 Phytochemical Study

221 Plant Material and Preparation of the Purified AqueousExtract Aerial parts of Acanthospermum hispidum were col-lected from the botanical garden of the Federal University ofGrande Dourados (UFGD Dourados Brazil) at 458m abovesea level (S 22∘11101584042710158401015840 and W 54∘56101584010210158401015840) in October 2015A voucher specimen was authenticated by Dra Maria doCarmo Vieira (DDMS number 5219) and deposited at theUFGD plant facility

A hispidum aqueous extract was obtained by infusion in asimilar manner to that popularly used in Brazil [6] andprepared according to Tirloni et al [5] For this A hispidumleaves were air-dried in an oven at 40∘C for 7 days and thenthe dried plant was cut and ground into powder form usingmechanical milling The extract was obtained by infusing1 liter of boiling water for each 60 grams of dried andpulverized plant Extraction was carried out until roomtemperature was reached (sim5 h) The infusion was treatedwith 3 volumes of EtOH which gave rise to a precipitateand an ethanol-soluble fraction (ESAH) ESAH was filteredethanol was totally removed by evaporation and the resulting

fraction was lyophilized (yield of 805) All preparationswere kept in freezer until further analyses

222 Sample Analysis (Liquid Chromatography-Mass Spec-trometry (LC-MS)) Chromatography was performed in anultra-performance liquid chromatography (UPLCWaters)using a reversed phase column HSS T3 C18 column with 100times 21mm and 17 120583m of particle size (Waters) with constanttemperature of 60∘C The solvents were ultra-pure water(Milli-Q Millipore) and acetonitrile (JT Baker) containing01 (vv) of formic acid 96 (Tedia) and the gradient wasperformed increasing the acetonitrile from0 to 10 in 6minthen to 80 in 14min at flow rate of 04mlminThe solventreturned to the initial condition in 15min and the system wasreequilibrated for 2min The sample was prepared at 2mgmL in MeOH-H2O (1 1 vv) with injections of 5120583L

The detection was provided by a high-resolution massspectrometer LTQOrbitrap XL (Thermo Scientific)The ionswere detected in the negative and positive modes The ionsource was held at 350∘C and the desolvation was aided bynitrogen stream at 40 arbitrary units in the sheath gas and10 au in the auxiliary gas Negative ions provided bestresults for the sample with the energies at 35 kV in thesource minus46V in the capillary and minus200V in the tube lensFragmentations were performed by higher-energy collisionaldissociation with normalized collision energy of 25ndash35 ForLC-MS analysis the MS resolution was set at 15000 FWHMmass accuracy which was obtained by external calibrationroutinely performed

23 Pharmacological Studies

231 Animals Twelve-week-old femaleWistar rats weighing200ndash300 gwere randomized and housed in plastic cages withenvironmental enrichment at 22 plusmn 2∘C under a 1212 h lightdark cycle 55 plusmn 10 humidity conditions with ad libitumaccess to food andwater All procedures were approved by theethical committee on animal use of the Federal University ofGrande Dourados (UFGD) (number 452016) and exper-iments were performed in accordance with internationalstandards and ethical guidelines on animal welfare

232 Ovariectomy and Induction of Renovascular Hyper-tension Rats were anesthetized with ketamine (100mgkg)and xylazine (20mgkg) by intraperitoneal route Afterovariectomy (OVT) renovascular hypertension was inducedusing the Goldblatt model (two kidneys one clip 2K1C) asdescribed by Umar et al [7]The left renal artery was exposedby retroperitoneal incision and dissected A silver clip (lumenof 022mm) was placed around the artery for partial occlu-sion In the Sham-operated group (placebo surgery) theartery was not clipped and ovaries were not removed Aftersurgery animals received saline for rehydration (2mlanimalsubcutaneously single administration) anti-inflammatory(indomethacin 2mgkg by oral route every 12 hours during3 days) and antibiotic (enrofloxacin 10mgkg subcuta-neously single dose) Systolic blood pressure (SBP) wasweekly measured (for 4 weeks) using the tail-cuff method

Evidence-Based Complementary and Alternative Medicine 3

Only hypertensive rats (SBP above 140mmHg) were used inexperiments

233 Experimental Groups Five weeks after surgery animalswere randomized and divided into 7 groups (119899 = 6-7) forhemodynamic and renal function studies Rats were treatedonce a day (by gavage) during 28 days with vehicle (2K1Cplus OVT = positive control group) hydrochlorothiazide(HCTZ 25mgkg standard diuretic drug) enalapril (ENAL15mgkg standard antihypertensive drug) or ESAH (30 100or 300mgkg) The Sham-operated group received vehicleunder the same conditions

234 Diuretic Activity The diuretic effects of ESAH wereaccessed according to methods previously described by Kauet al [8] with somemodifications [9]Weekly (on days 1 7 1421 and 28) rats were fasted overnight (6 h) with free accessto water Animals received an oral load of isotonic saline(09 NaCl 5ml100 g) to impose a controlled water andsalt balance before treatments Then rats were immediatelyplaced in metabolic cages Urine samples were collected ina graduated cylinder and the volume was recorded 8 hoursafter treatment (expressed as ml per 100 g body weight)During the experiment rats were food deprived pH anddensity were determined on fresh urine samples using digitalpH meter (Q400MT Quimis Instruments Brazil) and ahandheld refractometer (NO107 Nova Instruments Brazil)respectively Urinary sodium (Na+) potassium (K+) chloride(Clminus) and calcium (Ca++) levelswere quantified in automatedanalyzer (Roche Cobas Integra 400 plus) Excretion load(El) of Na+ K+ Clminus and Ca++ was obtained according tothe equation El = 119880119909 times 119881 where 119880119909 is the concentrationof electrolytes (mEqL) and 119881 is the urinary flow (mLmin)Results were expressed as 120583Eqmin100 g

235 Blood Pressure andHeart Rate Evaluation At the end ofthe trial period (day 28) rats were anesthetizedwith ketamine(100mgkg) and xylazine (20mgkg) by intramuscular routeImmediately a bolus injection of heparin (50 IU) was subcu-taneously applied The left carotid artery was cannulated andconnected to a pressure transducer coupled to a PowerLabrecording system and an application software (Chart v 41 allfrom ADI Instruments Castle Hill Australia) recordingsystolic blood pressure (SBP) and diastolic blood pressure(DBP) mean blood pressure (MBP) and heart rate (HR)After 15 minutes of stabilization changes in blood pressureand HR were recorded for 5min

236 Mesenteric Vascular Reactivity After systemic bloodpressure and HR measurements and before euthanasia themesenteric vascular bed (MVB) was isolated and preparedfor perfusion as previously described [10]The isolatedMVBswere then placed in awater-jacketed organ bathmaintained at37∘C and perfused with PSS (composition in mM NaCl 119KCl 47 CaCl2 24 MgSO4 12 NaHCO3 250 KH2PO4 12dextrose 111 and EDTA 003) gassed with 95 O25 CO2at constant flow rate of 4mlmin through a peristaltic pumpChanges in the perfusion pressure (mmHg) were measuredby a pressure transducer connected to an acquisition system

(PowerLab) and its application software (Chart v 71 bothfrom ADI Instruments Castle Hill Australia) After setupin the perfusion apparatus preparation was allowed toequilibrate for 30 to 45min and its viability was checked bya bolus injection of KCl (120mmol) After a 30-minute stabi-lization period a phenylephrine (PHE 3 10 and 30 nmol)dose-response administration was performed by injectingincreasing doses into the perfusion system After a 60-minute equilibration period MVBs were continuously per-fused with PSS containing 3 120583M PHE which was enough toinduce a sustained increased perfusion pressure Under theseconditions vasodilatory effects of acetylcholine (ACh 310 and 30 pmol) or sodium nitroprusside (SNP 3 10 and30 pmol) were measured A 15-minute equilibration periodwas allowed between each drug All drugs were given into theperfusate as bolus injection of 100 120583l

237 Serum Biochemical Parameters At the end of exper-iments all animals were killed by suprapharmacologicalisoflurane dose and blood samples were collected Serumwasobtained by centrifugation (1000timesg for 10min) Serum Na+K+ urea and creatinine were determined in automatedbiochemical analyzer (Roche Cobas Integra 400 plus)Serum nitrotyrosine (NT) aldosterone and vasopressinlevels were measured by enzyme-linked immunosorbentassay kit according to manufacturerrsquos specifications (BD Bio-sciences CA USA)Thiobarbituric acid (TBARS) levels weremeasured using TBARS assay kits (Cayman Chemical AnnArbor Michigan USA) according to manufacturerrsquos instruc-tion Plasma nitrite concentration was determined by enzy-matically reducing nitrate according to technique describedby Schmidt et al [11] Serum angiotensin converting enzyme(ACE) activity was determined by indirect fluorimetryaccording to methods described by Santos et al [12]

24 Investigation of the Molecular Mechanisms Involved in theVasodilatory Response of ESAH

241 Evaluation of the Effects of ESAH on the Mesenteric Vas-cular Bed Preparations (MVBs) with functional endothe-lium from normotensive and hypertensive (2K1C plus OVT)female rats without any previous treatment were contin-uously perfused with PSS containing PHE (3 120583M) Afterstabilization of the increased perfusion pressure preparationsreceived bolus injections containing 00003 0001 0003 and001mg of ESAH and the reduction in the perfusion pressurewas evaluated Each next dosewas administered only after thereturn of the perfusion pressure to the same level recordedbefore injection with minimal interval of 3min betweendoses

242 Investigation of the Mechanisms Involved in the VascularEffects of ESAH In these experiments after recording thefirst dose-response curve to ESAH (0001 0003 and001mg) the MVBs were left to equilibrate for an addi-tional period of 30ndash45min under perfusion with PSS Thendifferent preparations were perfused with PSS containing3 120583Mphenylephrine plus indomethacin (1 120583M a nonselective

4 Evidence-Based Complementary and Alternative Medicine

cyclooxygenase inhibitor) and L-NAME (100120583M a nons-elective NO synthase inhibitor) alone or combined After15min under perfusion with one of the previouslymentionedsolutions ESAH (0001 0003 and 001mg) was injectedagain into the perfusion systemTheability of ESAH to reducethe perfusion pressure was compared with results obtained incontrol preparations perfused with vehicle only

25 Statistical Analyses Data were analyzed for homogeneityof variance and normal distribution Differences betweenmeans were determined using one-way analysis of variance(ANOVA) followed by Dunnettrsquos post hoc test The level ofsignificance was set at 95 (119901 lt 005) and results areexpressed as mean plusmn standard error of the mean (SEM)Graphs were drawn and statistical analysis was carried outusing GraphPad Prism software version 50 for Mac OS X(GraphPad Software San Diego CA USA)

3 Results

31 Phytochemical Characterization In a previous work [6]the composition of an extract of Acanthospermum hispidumwas partially characterized with identification of the familyof monocaffeoylquinic acids (CQAs) and dicaffeoylquinicacids (diCQAs) observed as multiple isomers in the LC-MSanalysis However these isomers have characteristic frag-mentation profile that can be used for their identification[13 14] To confirm the isomer identity an extract of Ilexparaguariensis containing mono- and dicaffeoylquinic acidspreviously characterized [13] was used as a reference sample

The detailed chemical composition of ESAH obtainedfromA hispidum is shown in Table 1The negative ionizationwas better for the acidic compounds present in the extractand the monocaffeoylquinic acids appeared at 119898119911 353087The carbons from the ring of quinic acid were numeratedaccording to the chlorogenic acid structure considered as3-monocaffeoylquinic acid (3-CQA) Thus based on thefragmentation profile (at collision energy of 25) and retentiontime compared to I paraguariensis extract the isomers couldbe predicted as neo-chlorogenic acid (5-CQA at 230min)with intense fragments at 119898119911 191056 179035 and 135045but absent of 119898119911 17345 The chlorogenic acid (3-CQA)appeared at 322min with a main fragment at 119898119911 191056and the crypto-chlorogenic acid (4-CQA at 354min) pro-duced fragments at119898119911 191056 179035 173045 and 135045detaching the most abundant fragment-ion at 119898119911 173045(Figure 1) These results are in accordance with the pioneerwork of Carini et al [15]The peaks at 594 and 605min wereobserved at 119898119911 463088 with similar fragments at 119898119911300027 271024 255029 and 243029 consistent withquercetin-hexosides being consistent with quercetin galac-toside and quercetin glucoside [16]

The main peaks on chromatogram were identified asdicaffeoylquinic acids (di-CQAs) at 119898119911 515119 and similarto the CQAs they differ in their fragmentation profileobtained with collision energy at 35The peak at 643min wasconsistent with 34-di-CQA with fragments at 119898119911 353087due to the loss of a caffeoyl residue and the fragment at119898119911 335076 (353087 H2O) was observed at a relative

intensity in only this isomer The other fragments were 119898119911191056 (quinic acid base peak) 119898119911 179035 (caffeic acid)and 119898119911 173045 (quinic acid H2O) The peaks at 667and 707min presented a quite similar fragmentation profilecontaining almost all fragments observed for the peak at643min except by the absence of ion at 119898119911 335076 Thechromatographic profile suggests the peak at 667min as 35-di-CQA and the peak at 707min as 45-di-CQA Howeverother isomers may be eluted in very close retention time aspreviously observed [14] hindering the accurate isomer iden-tification Other peaks (728 754 and 852min) showed themain ion at 119898119911 337238 which could be result of in-sourcefragmentation and remains unidentified and the peaks at1038 1067 1077 and 1089min presented multiple ionsavoiding their identification

32 Prolonged Treatment with ESAH Induced SignificantSaluretic Effect on Sham-Operated and 2K1C Plus OVT-RatsThe values obtained for urinary volume and renal excretionof electrolytes are shown in Tables 2 3 4 5 and 6 The meanurinary volumes of Sham-operated rats collected for 8 hourson days 1 7 14 21 and 28 were respectively 564plusmn070 916plusmn109 1036 plusmn 132 898 plusmn 093 and 784 plusmn 055ml100 g8 hSimilarly 2K1C plus OVT-animals produced an estimatedurinary volume of 499 plusmn 027 556 plusmn 021 711 plusmn 062 716 plusmn042 and 592 plusmn 032ml100 g8 h on days 1 7 14 21 and 28respectively None of the groups treated with ENAL or ESAH(30 100 and 300mgkg) had any changes in urinary volumewhen compared to Sham-operated or 2K1C plus OVT-ratsAs expected animals that received HCTZ had a significantincrease in urinary volume only on the 1st day of treatment

On the 1st day of treatment all animals treatedwith ESAH(30 100 or 300mgkg) or HCTZ presented a significantincrease in Na+ and Clminus elimination with saluretic indexhigher than 20 (Table 2) On the other hand only animalstreated with ESAH (30 100 and 300mgkg) maintained thiseffect on the 7th day of treatment (Table 3) On the 14th 21stand 28th days of treatment only animals receiving ESAH atthe dose of 300mgkg had significant urinary Na+ and Clminuslevels (Tables 4 5 and 6) In contrast animals treated withHCTZ returned to significant Na+ levels only on the 28thday of treatment (Table 6) Groups receiving ENAL did notshow any significant increase in urinaryNa+ K+ Clminus or Ca++when compared with positive controls or Sham-operatedanimals Similarly urinary pH and density or serumNa+ K+urea and creatinine values were not altered by any of thetreatments (Tables 2 3 4 5 6 and 7)

33 ESAH Administration Reduces SBP DBP MBP and HRin 2K1C Plus OVT-Rats Figures 2(a) 2(b) 2(c) and 2(d)show the systemic blood pressure and HR values obtainedafter prolonged treatment with ESAH ENAL or HCTZ Allanimals in the positive control group presented significantincrease in SBP (144 plusmn 53 versus 111 plusmn 37mmHg) DBP(103plusmn28 versus 82plusmn29mmHg) MBP (110plusmn38 versus 92plusmn28mmHg) and HR values (251 plusmn 12 versus 202 plusmn 46 bpm)when compared to Sham-operated rats Prolonged adminis-tration of ESAH at doses of 30mgkg was able to reduce SBPDBP and MBP to 90 plusmn 55 71 plusmn 44 and 77 plusmn 55mmHg

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Phytochemical composition of ESAH obtained from A hispidum DC

Peak Rt (min) MS1 MS2 (key fragments) Tentative identification1 230 35308773 [M-H]minus 191055 179034 135045 neo-Chlorogenic acid2 322 35308745 [M-H]minus 191055 Chlorogenic acid3 354 35308773 [M-H]minus 191055 179034 173045 135044 crypto-Chlorogenic acid4 594 46308838 [M-H]minus 300027 Quercetin-galactoside5 605 46308804 [M-H]minus 300027 Quercetin-glucoside6 643 51511964 [M-H]minus 353087 335076 191055 179034 173045 135045 34-Dicaffeoylquinic acid7 667 51511970 [M-H]minus 353087 191055 179034173045 135044 35-Dicaffeoylquinic acid8 707 51511951 [M-H]minus 353087 191055 179034173045 135044 45-Dicaffeoylquinic acid9 728 53526876 [M+Cl]minus 337238 C26H44O9Cl10 754 33723821 [M-H]minus mdash C20H33O411 852 33723840 [M-H]minus mdash C20H33O4

Table 2 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 1st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 564 plusmn 070 037 plusmn 006 049 plusmn 006 046 plusmn 003 0036 plusmn 00100 796 plusmn 015 1016 plusmn 189

C+ 499 plusmn 027 036 plusmn 002 044 plusmn 003 042 plusmn 002 0046 plusmn 00058 791 plusmn 007 1019 plusmn 066

ESAH (30mgkg) 381 plusmn 046 106 plusmn 007ab 067 plusmn 006 122 plusmn 008ab 0033 plusmn 00068 782 plusmn 019 1014 plusmn 223

ESAH (100mgkg) 396 plusmn 036 092 plusmn 019ab 053 plusmn 007 108 plusmn 023ab 0034 plusmn 00041 781 plusmn 013 1019 plusmn 210

ESAH (300mgkg) 344 plusmn 026 080 plusmn 012ab 049 plusmn 007 100 plusmn 008ab 0036 plusmn 00068 743 plusmn 014 1018 plusmn 176

ENAL 472 plusmn 059 027 plusmn 005 055 plusmn 003 037 plusmn 006 0041 plusmn 00019 796 plusmn 015 1017 plusmn 097

HCTZ 761 plusmn 028a 099 plusmn 024ab 075 plusmn 017 118 plusmn 030ab 0030 plusmn 00069 780 plusmn 013 1015 plusmn 117

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

respectively whileHR levels were reduced to 165plusmn77 bpm Inaddition ESAH at doses of 100 and 300mg was not ableto change the SBP DBP MBP or HR of normotensive or2K1CplusOVT-ratsHowever as expected ENAL andHCTZwere able to significantly reduce SBP DBP andMBP levels inpositive controls or Sham-operated animals Values obtainedwith ENAL or HCTZ administration on the arterial pressurelevels were not statistically different from those observed aftertreatment with ESAH (30mgkg)

34 Prolonged Treatment with ESAH Restores Vascular Reac-tivity in MVBs from 2K1C OVT-Rats In MVBs from 2K1Cplus OVT-rats the administration of ACh or SNP was able toinduce a vasodilatory response approximately 35 lower thanthat in Sham-operated rats (Figures 3(a) and 3(b)) Similarlythe vasoconstrictor response of PHE was significantly higherin the 2K1C plus OVT-group when compared to Sham-operated animals (Figure 3(c)) On the other hand in animalsthat received ESAH (30mgkg) or ENAL (15mgkg) theeffects of ACh SNP or PHE were not different from thoseobserved in Sham-operated animals Treatments with HCTZ(25mgkg) or ESAH at doses of 100 and 300mgkg were notable to reverse the changes in vascular reactivity observed in2K1C plus OVT-animals (Figures 3(a)ndash3(c))

35 ESAH Reduces Oxidative and Nitrosative Stress withoutAffectingAldosterone Vasopressin Levels or SerumACEActiv-ity The effects of ESAH ENAL and HCTZ on TBARS NT

and nitrite levels are shown in Figures 4(a) 4(b) and4(c) Ovariectomy associatedwith renovascular hypertensionincreases TBARS andNT levels insim104 and 60 respectivelyTreatment with ESAH (30 100 and 300mgkg) or ENALreduced TBARS and NT levels to values close to those foundin Sham-operated animals

At baseline nitrite concentration was significantly lowerin positive control animals when compared with Sham-operated ones (63 plusmn 53 versus 94 plusmn 50 120583M) On the otherhand treatment with ESAH (at all doses) or ENAL increasedthe nitrite levels by sim50

On the other hand only ENAL was able to reducethe aldosterone levels and serum ACE activity (Figures4(a)ndash4(c)) without affecting serum vasopressin Treatmentwith ESAH (30 100 and 300mgkg) orHCTZdid not signifi-cantly change the aldosterone and vasopressin concentrationsor serum ACE activity (Figures 5(a)ndash5(c))

36 ESAH Induce Vasodilatory Response in MVBs fromNormotensive and 2K1C Plus OVT-Rats in Dependence onEndothelial NO and Prostaglandins The continuous perfu-sion of the MVBs (from normotensive or 2K1C plus OVT-rats) with phenylephrine resulted in a sustained increase inthe vascular perfusion pressure which was significantlyreduced by ESAH (0001 0003 and 001mg) (Figures 6(b)and 6(d))The characterization of a typical experiment shownin Figure 6(a) (normotensive) or Figure 6(c) (2K1C plusOVT-rats) reveals that when the highest ESAH dose was

6 Evidence-Based Complementary and Alternative Medicine

0102030405060708090

100

Rela

tive a

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ance

038 041095 188 230 255

322354 423 494 587

594

643

667

728754

761825

852

872 913 946 10381067

1077

10891178 1241 1279 1311 1372 1435

708

179035 HO HO

HOHO

HO

HO

HO

HO

HO

O

O

O

O

O

OO

O

O

O

O

O

O OOH OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

191056

191056

353087

353087

353087191056

191056

191056

191056

179035

179035

179035

179035

135045135045

173045

173045

335077 335077

353087 353087191056

135045135045 135045335077

353087353087

353087

515119 515119 515119

173045

173045 173045179035

179035 179035

191056

191056191056

0102030405060708090

100Re

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e abu

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ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150Time (min)

0102030405060708090

100

Rela

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OminusOminus

Ominus

Ominus

350250 300200150mz

350250 300200150mz

350250 300200150mz

0102030405060708090

100

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0102030405060708090

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Rela

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200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

0102030405060708090

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0102030405060708090

100

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tive a

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(a)

(b) (c) (d)

(e) (f) (g)

Figure 1 Phytochemical analysis of ESAH obtained from A hispidum DC (a) Base peak chromatogram obtained by UPLC detected byHR-MS in the negative ionization and the fragmentation profile of monocaffeoylquinic acids tentatively identified as neo-chlorogenic acid(5-CQA) (b) chlorogenic acid (3-CQA) (c) and crypto-chlorogenic acid (4-CQA) (d) The main breakdown site for dicaffeoylquinic acids isdepicted in the 35 dicaffeoylquinic acid (35-di-CQA) and the fragmentation profile observed for peak 643min (e) 667min (f) and 707min(g)

used (001mg) the vasodilatory effects of ESAHhad responseintensity similar to that of acetylcholine

Previous infusion with L-NAME significantly reducedpart of the effect on theMVBs obtained with ESAHThe peakeffect of 0001 0003 and 001mg of ESAH was decreasedfrom 28 plusmn 4 54 plusmn 6 and 68 plusmn 7 to 11 plusmn 3 22 plusmn 9 and

26 plusmn 11 in normotensive and from 24 plusmn 3 50 plusmn 7 and62 plusmn 9 to 9 plusmn 2 18 plusmn 7 and 16 plusmn 6 in 2K1C plus OVT-rats respectively (Figures 7(a) and 7(d)) In a similar waythe vasodilatory effect of ESAH at doses of 0001 0003 and001mg was reduced to 20 plusmn 7 25 plusmn 8 and 27 plusmn 9 innormotensive and to 14 plusmn 3 21 plusmn 6 and 20 plusmn 7 in 2K1C

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

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Evidence-Based Complementary and Alternative Medicine 3

Only hypertensive rats (SBP above 140mmHg) were used inexperiments

233 Experimental Groups Five weeks after surgery animalswere randomized and divided into 7 groups (119899 = 6-7) forhemodynamic and renal function studies Rats were treatedonce a day (by gavage) during 28 days with vehicle (2K1Cplus OVT = positive control group) hydrochlorothiazide(HCTZ 25mgkg standard diuretic drug) enalapril (ENAL15mgkg standard antihypertensive drug) or ESAH (30 100or 300mgkg) The Sham-operated group received vehicleunder the same conditions

234 Diuretic Activity The diuretic effects of ESAH wereaccessed according to methods previously described by Kauet al [8] with somemodifications [9]Weekly (on days 1 7 1421 and 28) rats were fasted overnight (6 h) with free accessto water Animals received an oral load of isotonic saline(09 NaCl 5ml100 g) to impose a controlled water andsalt balance before treatments Then rats were immediatelyplaced in metabolic cages Urine samples were collected ina graduated cylinder and the volume was recorded 8 hoursafter treatment (expressed as ml per 100 g body weight)During the experiment rats were food deprived pH anddensity were determined on fresh urine samples using digitalpH meter (Q400MT Quimis Instruments Brazil) and ahandheld refractometer (NO107 Nova Instruments Brazil)respectively Urinary sodium (Na+) potassium (K+) chloride(Clminus) and calcium (Ca++) levelswere quantified in automatedanalyzer (Roche Cobas Integra 400 plus) Excretion load(El) of Na+ K+ Clminus and Ca++ was obtained according tothe equation El = 119880119909 times 119881 where 119880119909 is the concentrationof electrolytes (mEqL) and 119881 is the urinary flow (mLmin)Results were expressed as 120583Eqmin100 g

235 Blood Pressure andHeart Rate Evaluation At the end ofthe trial period (day 28) rats were anesthetizedwith ketamine(100mgkg) and xylazine (20mgkg) by intramuscular routeImmediately a bolus injection of heparin (50 IU) was subcu-taneously applied The left carotid artery was cannulated andconnected to a pressure transducer coupled to a PowerLabrecording system and an application software (Chart v 41 allfrom ADI Instruments Castle Hill Australia) recordingsystolic blood pressure (SBP) and diastolic blood pressure(DBP) mean blood pressure (MBP) and heart rate (HR)After 15 minutes of stabilization changes in blood pressureand HR were recorded for 5min

236 Mesenteric Vascular Reactivity After systemic bloodpressure and HR measurements and before euthanasia themesenteric vascular bed (MVB) was isolated and preparedfor perfusion as previously described [10]The isolatedMVBswere then placed in awater-jacketed organ bathmaintained at37∘C and perfused with PSS (composition in mM NaCl 119KCl 47 CaCl2 24 MgSO4 12 NaHCO3 250 KH2PO4 12dextrose 111 and EDTA 003) gassed with 95 O25 CO2at constant flow rate of 4mlmin through a peristaltic pumpChanges in the perfusion pressure (mmHg) were measuredby a pressure transducer connected to an acquisition system

(PowerLab) and its application software (Chart v 71 bothfrom ADI Instruments Castle Hill Australia) After setupin the perfusion apparatus preparation was allowed toequilibrate for 30 to 45min and its viability was checked bya bolus injection of KCl (120mmol) After a 30-minute stabi-lization period a phenylephrine (PHE 3 10 and 30 nmol)dose-response administration was performed by injectingincreasing doses into the perfusion system After a 60-minute equilibration period MVBs were continuously per-fused with PSS containing 3 120583M PHE which was enough toinduce a sustained increased perfusion pressure Under theseconditions vasodilatory effects of acetylcholine (ACh 310 and 30 pmol) or sodium nitroprusside (SNP 3 10 and30 pmol) were measured A 15-minute equilibration periodwas allowed between each drug All drugs were given into theperfusate as bolus injection of 100 120583l

237 Serum Biochemical Parameters At the end of exper-iments all animals were killed by suprapharmacologicalisoflurane dose and blood samples were collected Serumwasobtained by centrifugation (1000timesg for 10min) Serum Na+K+ urea and creatinine were determined in automatedbiochemical analyzer (Roche Cobas Integra 400 plus)Serum nitrotyrosine (NT) aldosterone and vasopressinlevels were measured by enzyme-linked immunosorbentassay kit according to manufacturerrsquos specifications (BD Bio-sciences CA USA)Thiobarbituric acid (TBARS) levels weremeasured using TBARS assay kits (Cayman Chemical AnnArbor Michigan USA) according to manufacturerrsquos instruc-tion Plasma nitrite concentration was determined by enzy-matically reducing nitrate according to technique describedby Schmidt et al [11] Serum angiotensin converting enzyme(ACE) activity was determined by indirect fluorimetryaccording to methods described by Santos et al [12]

24 Investigation of the Molecular Mechanisms Involved in theVasodilatory Response of ESAH

241 Evaluation of the Effects of ESAH on the Mesenteric Vas-cular Bed Preparations (MVBs) with functional endothe-lium from normotensive and hypertensive (2K1C plus OVT)female rats without any previous treatment were contin-uously perfused with PSS containing PHE (3 120583M) Afterstabilization of the increased perfusion pressure preparationsreceived bolus injections containing 00003 0001 0003 and001mg of ESAH and the reduction in the perfusion pressurewas evaluated Each next dosewas administered only after thereturn of the perfusion pressure to the same level recordedbefore injection with minimal interval of 3min betweendoses

242 Investigation of the Mechanisms Involved in the VascularEffects of ESAH In these experiments after recording thefirst dose-response curve to ESAH (0001 0003 and001mg) the MVBs were left to equilibrate for an addi-tional period of 30ndash45min under perfusion with PSS Thendifferent preparations were perfused with PSS containing3 120583Mphenylephrine plus indomethacin (1 120583M a nonselective

4 Evidence-Based Complementary and Alternative Medicine

cyclooxygenase inhibitor) and L-NAME (100120583M a nons-elective NO synthase inhibitor) alone or combined After15min under perfusion with one of the previouslymentionedsolutions ESAH (0001 0003 and 001mg) was injectedagain into the perfusion systemTheability of ESAH to reducethe perfusion pressure was compared with results obtained incontrol preparations perfused with vehicle only

25 Statistical Analyses Data were analyzed for homogeneityof variance and normal distribution Differences betweenmeans were determined using one-way analysis of variance(ANOVA) followed by Dunnettrsquos post hoc test The level ofsignificance was set at 95 (119901 lt 005) and results areexpressed as mean plusmn standard error of the mean (SEM)Graphs were drawn and statistical analysis was carried outusing GraphPad Prism software version 50 for Mac OS X(GraphPad Software San Diego CA USA)

3 Results

31 Phytochemical Characterization In a previous work [6]the composition of an extract of Acanthospermum hispidumwas partially characterized with identification of the familyof monocaffeoylquinic acids (CQAs) and dicaffeoylquinicacids (diCQAs) observed as multiple isomers in the LC-MSanalysis However these isomers have characteristic frag-mentation profile that can be used for their identification[13 14] To confirm the isomer identity an extract of Ilexparaguariensis containing mono- and dicaffeoylquinic acidspreviously characterized [13] was used as a reference sample

The detailed chemical composition of ESAH obtainedfromA hispidum is shown in Table 1The negative ionizationwas better for the acidic compounds present in the extractand the monocaffeoylquinic acids appeared at 119898119911 353087The carbons from the ring of quinic acid were numeratedaccording to the chlorogenic acid structure considered as3-monocaffeoylquinic acid (3-CQA) Thus based on thefragmentation profile (at collision energy of 25) and retentiontime compared to I paraguariensis extract the isomers couldbe predicted as neo-chlorogenic acid (5-CQA at 230min)with intense fragments at 119898119911 191056 179035 and 135045but absent of 119898119911 17345 The chlorogenic acid (3-CQA)appeared at 322min with a main fragment at 119898119911 191056and the crypto-chlorogenic acid (4-CQA at 354min) pro-duced fragments at119898119911 191056 179035 173045 and 135045detaching the most abundant fragment-ion at 119898119911 173045(Figure 1) These results are in accordance with the pioneerwork of Carini et al [15]The peaks at 594 and 605min wereobserved at 119898119911 463088 with similar fragments at 119898119911300027 271024 255029 and 243029 consistent withquercetin-hexosides being consistent with quercetin galac-toside and quercetin glucoside [16]

The main peaks on chromatogram were identified asdicaffeoylquinic acids (di-CQAs) at 119898119911 515119 and similarto the CQAs they differ in their fragmentation profileobtained with collision energy at 35The peak at 643min wasconsistent with 34-di-CQA with fragments at 119898119911 353087due to the loss of a caffeoyl residue and the fragment at119898119911 335076 (353087 H2O) was observed at a relative

intensity in only this isomer The other fragments were 119898119911191056 (quinic acid base peak) 119898119911 179035 (caffeic acid)and 119898119911 173045 (quinic acid H2O) The peaks at 667and 707min presented a quite similar fragmentation profilecontaining almost all fragments observed for the peak at643min except by the absence of ion at 119898119911 335076 Thechromatographic profile suggests the peak at 667min as 35-di-CQA and the peak at 707min as 45-di-CQA Howeverother isomers may be eluted in very close retention time aspreviously observed [14] hindering the accurate isomer iden-tification Other peaks (728 754 and 852min) showed themain ion at 119898119911 337238 which could be result of in-sourcefragmentation and remains unidentified and the peaks at1038 1067 1077 and 1089min presented multiple ionsavoiding their identification

32 Prolonged Treatment with ESAH Induced SignificantSaluretic Effect on Sham-Operated and 2K1C Plus OVT-RatsThe values obtained for urinary volume and renal excretionof electrolytes are shown in Tables 2 3 4 5 and 6 The meanurinary volumes of Sham-operated rats collected for 8 hourson days 1 7 14 21 and 28 were respectively 564plusmn070 916plusmn109 1036 plusmn 132 898 plusmn 093 and 784 plusmn 055ml100 g8 hSimilarly 2K1C plus OVT-animals produced an estimatedurinary volume of 499 plusmn 027 556 plusmn 021 711 plusmn 062 716 plusmn042 and 592 plusmn 032ml100 g8 h on days 1 7 14 21 and 28respectively None of the groups treated with ENAL or ESAH(30 100 and 300mgkg) had any changes in urinary volumewhen compared to Sham-operated or 2K1C plus OVT-ratsAs expected animals that received HCTZ had a significantincrease in urinary volume only on the 1st day of treatment

On the 1st day of treatment all animals treatedwith ESAH(30 100 or 300mgkg) or HCTZ presented a significantincrease in Na+ and Clminus elimination with saluretic indexhigher than 20 (Table 2) On the other hand only animalstreated with ESAH (30 100 and 300mgkg) maintained thiseffect on the 7th day of treatment (Table 3) On the 14th 21stand 28th days of treatment only animals receiving ESAH atthe dose of 300mgkg had significant urinary Na+ and Clminuslevels (Tables 4 5 and 6) In contrast animals treated withHCTZ returned to significant Na+ levels only on the 28thday of treatment (Table 6) Groups receiving ENAL did notshow any significant increase in urinaryNa+ K+ Clminus or Ca++when compared with positive controls or Sham-operatedanimals Similarly urinary pH and density or serumNa+ K+urea and creatinine values were not altered by any of thetreatments (Tables 2 3 4 5 6 and 7)

33 ESAH Administration Reduces SBP DBP MBP and HRin 2K1C Plus OVT-Rats Figures 2(a) 2(b) 2(c) and 2(d)show the systemic blood pressure and HR values obtainedafter prolonged treatment with ESAH ENAL or HCTZ Allanimals in the positive control group presented significantincrease in SBP (144 plusmn 53 versus 111 plusmn 37mmHg) DBP(103plusmn28 versus 82plusmn29mmHg) MBP (110plusmn38 versus 92plusmn28mmHg) and HR values (251 plusmn 12 versus 202 plusmn 46 bpm)when compared to Sham-operated rats Prolonged adminis-tration of ESAH at doses of 30mgkg was able to reduce SBPDBP and MBP to 90 plusmn 55 71 plusmn 44 and 77 plusmn 55mmHg

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Phytochemical composition of ESAH obtained from A hispidum DC

Peak Rt (min) MS1 MS2 (key fragments) Tentative identification1 230 35308773 [M-H]minus 191055 179034 135045 neo-Chlorogenic acid2 322 35308745 [M-H]minus 191055 Chlorogenic acid3 354 35308773 [M-H]minus 191055 179034 173045 135044 crypto-Chlorogenic acid4 594 46308838 [M-H]minus 300027 Quercetin-galactoside5 605 46308804 [M-H]minus 300027 Quercetin-glucoside6 643 51511964 [M-H]minus 353087 335076 191055 179034 173045 135045 34-Dicaffeoylquinic acid7 667 51511970 [M-H]minus 353087 191055 179034173045 135044 35-Dicaffeoylquinic acid8 707 51511951 [M-H]minus 353087 191055 179034173045 135044 45-Dicaffeoylquinic acid9 728 53526876 [M+Cl]minus 337238 C26H44O9Cl10 754 33723821 [M-H]minus mdash C20H33O411 852 33723840 [M-H]minus mdash C20H33O4

Table 2 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 1st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 564 plusmn 070 037 plusmn 006 049 plusmn 006 046 plusmn 003 0036 plusmn 00100 796 plusmn 015 1016 plusmn 189

C+ 499 plusmn 027 036 plusmn 002 044 plusmn 003 042 plusmn 002 0046 plusmn 00058 791 plusmn 007 1019 plusmn 066

ESAH (30mgkg) 381 plusmn 046 106 plusmn 007ab 067 plusmn 006 122 plusmn 008ab 0033 plusmn 00068 782 plusmn 019 1014 plusmn 223

ESAH (100mgkg) 396 plusmn 036 092 plusmn 019ab 053 plusmn 007 108 plusmn 023ab 0034 plusmn 00041 781 plusmn 013 1019 plusmn 210

ESAH (300mgkg) 344 plusmn 026 080 plusmn 012ab 049 plusmn 007 100 plusmn 008ab 0036 plusmn 00068 743 plusmn 014 1018 plusmn 176

ENAL 472 plusmn 059 027 plusmn 005 055 plusmn 003 037 plusmn 006 0041 plusmn 00019 796 plusmn 015 1017 plusmn 097

HCTZ 761 plusmn 028a 099 plusmn 024ab 075 plusmn 017 118 plusmn 030ab 0030 plusmn 00069 780 plusmn 013 1015 plusmn 117

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

respectively whileHR levels were reduced to 165plusmn77 bpm Inaddition ESAH at doses of 100 and 300mg was not ableto change the SBP DBP MBP or HR of normotensive or2K1CplusOVT-ratsHowever as expected ENAL andHCTZwere able to significantly reduce SBP DBP andMBP levels inpositive controls or Sham-operated animals Values obtainedwith ENAL or HCTZ administration on the arterial pressurelevels were not statistically different from those observed aftertreatment with ESAH (30mgkg)

34 Prolonged Treatment with ESAH Restores Vascular Reac-tivity in MVBs from 2K1C OVT-Rats In MVBs from 2K1Cplus OVT-rats the administration of ACh or SNP was able toinduce a vasodilatory response approximately 35 lower thanthat in Sham-operated rats (Figures 3(a) and 3(b)) Similarlythe vasoconstrictor response of PHE was significantly higherin the 2K1C plus OVT-group when compared to Sham-operated animals (Figure 3(c)) On the other hand in animalsthat received ESAH (30mgkg) or ENAL (15mgkg) theeffects of ACh SNP or PHE were not different from thoseobserved in Sham-operated animals Treatments with HCTZ(25mgkg) or ESAH at doses of 100 and 300mgkg were notable to reverse the changes in vascular reactivity observed in2K1C plus OVT-animals (Figures 3(a)ndash3(c))

35 ESAH Reduces Oxidative and Nitrosative Stress withoutAffectingAldosterone Vasopressin Levels or SerumACEActiv-ity The effects of ESAH ENAL and HCTZ on TBARS NT

and nitrite levels are shown in Figures 4(a) 4(b) and4(c) Ovariectomy associatedwith renovascular hypertensionincreases TBARS andNT levels insim104 and 60 respectivelyTreatment with ESAH (30 100 and 300mgkg) or ENALreduced TBARS and NT levels to values close to those foundin Sham-operated animals

At baseline nitrite concentration was significantly lowerin positive control animals when compared with Sham-operated ones (63 plusmn 53 versus 94 plusmn 50 120583M) On the otherhand treatment with ESAH (at all doses) or ENAL increasedthe nitrite levels by sim50

On the other hand only ENAL was able to reducethe aldosterone levels and serum ACE activity (Figures4(a)ndash4(c)) without affecting serum vasopressin Treatmentwith ESAH (30 100 and 300mgkg) orHCTZdid not signifi-cantly change the aldosterone and vasopressin concentrationsor serum ACE activity (Figures 5(a)ndash5(c))

36 ESAH Induce Vasodilatory Response in MVBs fromNormotensive and 2K1C Plus OVT-Rats in Dependence onEndothelial NO and Prostaglandins The continuous perfu-sion of the MVBs (from normotensive or 2K1C plus OVT-rats) with phenylephrine resulted in a sustained increase inthe vascular perfusion pressure which was significantlyreduced by ESAH (0001 0003 and 001mg) (Figures 6(b)and 6(d))The characterization of a typical experiment shownin Figure 6(a) (normotensive) or Figure 6(c) (2K1C plusOVT-rats) reveals that when the highest ESAH dose was

6 Evidence-Based Complementary and Alternative Medicine

0102030405060708090

100

Rela

tive a

bund

ance

038 041095 188 230 255

322354 423 494 587

594

643

667

728754

761825

852

872 913 946 10381067

1077

10891178 1241 1279 1311 1372 1435

708

179035 HO HO

HOHO

HO

HO

HO

HO

HO

O

O

O

O

O

OO

O

O

O

O

O

O OOH OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

191056

191056

353087

353087

353087191056

191056

191056

191056

179035

179035

179035

179035

135045135045

173045

173045

335077 335077

353087 353087191056

135045135045 135045335077

353087353087

353087

515119 515119 515119

173045

173045 173045179035

179035 179035

191056

191056191056

0102030405060708090

100Re

lativ

e abu

ndan

ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150Time (min)

0102030405060708090

100

Rela

tive a

bund

ance

OminusOminus

Ominus

Ominus

350250 300200150mz

350250 300200150mz

350250 300200150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

(a)

(b) (c) (d)

(e) (f) (g)

Figure 1 Phytochemical analysis of ESAH obtained from A hispidum DC (a) Base peak chromatogram obtained by UPLC detected byHR-MS in the negative ionization and the fragmentation profile of monocaffeoylquinic acids tentatively identified as neo-chlorogenic acid(5-CQA) (b) chlorogenic acid (3-CQA) (c) and crypto-chlorogenic acid (4-CQA) (d) The main breakdown site for dicaffeoylquinic acids isdepicted in the 35 dicaffeoylquinic acid (35-di-CQA) and the fragmentation profile observed for peak 643min (e) 667min (f) and 707min(g)

used (001mg) the vasodilatory effects of ESAHhad responseintensity similar to that of acetylcholine

Previous infusion with L-NAME significantly reducedpart of the effect on theMVBs obtained with ESAHThe peakeffect of 0001 0003 and 001mg of ESAH was decreasedfrom 28 plusmn 4 54 plusmn 6 and 68 plusmn 7 to 11 plusmn 3 22 plusmn 9 and

26 plusmn 11 in normotensive and from 24 plusmn 3 50 plusmn 7 and62 plusmn 9 to 9 plusmn 2 18 plusmn 7 and 16 plusmn 6 in 2K1C plus OVT-rats respectively (Figures 7(a) and 7(d)) In a similar waythe vasodilatory effect of ESAH at doses of 0001 0003 and001mg was reduced to 20 plusmn 7 25 plusmn 8 and 27 plusmn 9 innormotensive and to 14 plusmn 3 21 plusmn 6 and 20 plusmn 7 in 2K1C

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Evidence-Based Complementary and Alternative Medicine

cyclooxygenase inhibitor) and L-NAME (100120583M a nons-elective NO synthase inhibitor) alone or combined After15min under perfusion with one of the previouslymentionedsolutions ESAH (0001 0003 and 001mg) was injectedagain into the perfusion systemTheability of ESAH to reducethe perfusion pressure was compared with results obtained incontrol preparations perfused with vehicle only

25 Statistical Analyses Data were analyzed for homogeneityof variance and normal distribution Differences betweenmeans were determined using one-way analysis of variance(ANOVA) followed by Dunnettrsquos post hoc test The level ofsignificance was set at 95 (119901 lt 005) and results areexpressed as mean plusmn standard error of the mean (SEM)Graphs were drawn and statistical analysis was carried outusing GraphPad Prism software version 50 for Mac OS X(GraphPad Software San Diego CA USA)

3 Results

31 Phytochemical Characterization In a previous work [6]the composition of an extract of Acanthospermum hispidumwas partially characterized with identification of the familyof monocaffeoylquinic acids (CQAs) and dicaffeoylquinicacids (diCQAs) observed as multiple isomers in the LC-MSanalysis However these isomers have characteristic frag-mentation profile that can be used for their identification[13 14] To confirm the isomer identity an extract of Ilexparaguariensis containing mono- and dicaffeoylquinic acidspreviously characterized [13] was used as a reference sample

The detailed chemical composition of ESAH obtainedfromA hispidum is shown in Table 1The negative ionizationwas better for the acidic compounds present in the extractand the monocaffeoylquinic acids appeared at 119898119911 353087The carbons from the ring of quinic acid were numeratedaccording to the chlorogenic acid structure considered as3-monocaffeoylquinic acid (3-CQA) Thus based on thefragmentation profile (at collision energy of 25) and retentiontime compared to I paraguariensis extract the isomers couldbe predicted as neo-chlorogenic acid (5-CQA at 230min)with intense fragments at 119898119911 191056 179035 and 135045but absent of 119898119911 17345 The chlorogenic acid (3-CQA)appeared at 322min with a main fragment at 119898119911 191056and the crypto-chlorogenic acid (4-CQA at 354min) pro-duced fragments at119898119911 191056 179035 173045 and 135045detaching the most abundant fragment-ion at 119898119911 173045(Figure 1) These results are in accordance with the pioneerwork of Carini et al [15]The peaks at 594 and 605min wereobserved at 119898119911 463088 with similar fragments at 119898119911300027 271024 255029 and 243029 consistent withquercetin-hexosides being consistent with quercetin galac-toside and quercetin glucoside [16]

The main peaks on chromatogram were identified asdicaffeoylquinic acids (di-CQAs) at 119898119911 515119 and similarto the CQAs they differ in their fragmentation profileobtained with collision energy at 35The peak at 643min wasconsistent with 34-di-CQA with fragments at 119898119911 353087due to the loss of a caffeoyl residue and the fragment at119898119911 335076 (353087 H2O) was observed at a relative

intensity in only this isomer The other fragments were 119898119911191056 (quinic acid base peak) 119898119911 179035 (caffeic acid)and 119898119911 173045 (quinic acid H2O) The peaks at 667and 707min presented a quite similar fragmentation profilecontaining almost all fragments observed for the peak at643min except by the absence of ion at 119898119911 335076 Thechromatographic profile suggests the peak at 667min as 35-di-CQA and the peak at 707min as 45-di-CQA Howeverother isomers may be eluted in very close retention time aspreviously observed [14] hindering the accurate isomer iden-tification Other peaks (728 754 and 852min) showed themain ion at 119898119911 337238 which could be result of in-sourcefragmentation and remains unidentified and the peaks at1038 1067 1077 and 1089min presented multiple ionsavoiding their identification

32 Prolonged Treatment with ESAH Induced SignificantSaluretic Effect on Sham-Operated and 2K1C Plus OVT-RatsThe values obtained for urinary volume and renal excretionof electrolytes are shown in Tables 2 3 4 5 and 6 The meanurinary volumes of Sham-operated rats collected for 8 hourson days 1 7 14 21 and 28 were respectively 564plusmn070 916plusmn109 1036 plusmn 132 898 plusmn 093 and 784 plusmn 055ml100 g8 hSimilarly 2K1C plus OVT-animals produced an estimatedurinary volume of 499 plusmn 027 556 plusmn 021 711 plusmn 062 716 plusmn042 and 592 plusmn 032ml100 g8 h on days 1 7 14 21 and 28respectively None of the groups treated with ENAL or ESAH(30 100 and 300mgkg) had any changes in urinary volumewhen compared to Sham-operated or 2K1C plus OVT-ratsAs expected animals that received HCTZ had a significantincrease in urinary volume only on the 1st day of treatment

On the 1st day of treatment all animals treatedwith ESAH(30 100 or 300mgkg) or HCTZ presented a significantincrease in Na+ and Clminus elimination with saluretic indexhigher than 20 (Table 2) On the other hand only animalstreated with ESAH (30 100 and 300mgkg) maintained thiseffect on the 7th day of treatment (Table 3) On the 14th 21stand 28th days of treatment only animals receiving ESAH atthe dose of 300mgkg had significant urinary Na+ and Clminuslevels (Tables 4 5 and 6) In contrast animals treated withHCTZ returned to significant Na+ levels only on the 28thday of treatment (Table 6) Groups receiving ENAL did notshow any significant increase in urinaryNa+ K+ Clminus or Ca++when compared with positive controls or Sham-operatedanimals Similarly urinary pH and density or serumNa+ K+urea and creatinine values were not altered by any of thetreatments (Tables 2 3 4 5 6 and 7)

33 ESAH Administration Reduces SBP DBP MBP and HRin 2K1C Plus OVT-Rats Figures 2(a) 2(b) 2(c) and 2(d)show the systemic blood pressure and HR values obtainedafter prolonged treatment with ESAH ENAL or HCTZ Allanimals in the positive control group presented significantincrease in SBP (144 plusmn 53 versus 111 plusmn 37mmHg) DBP(103plusmn28 versus 82plusmn29mmHg) MBP (110plusmn38 versus 92plusmn28mmHg) and HR values (251 plusmn 12 versus 202 plusmn 46 bpm)when compared to Sham-operated rats Prolonged adminis-tration of ESAH at doses of 30mgkg was able to reduce SBPDBP and MBP to 90 plusmn 55 71 plusmn 44 and 77 plusmn 55mmHg

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Phytochemical composition of ESAH obtained from A hispidum DC

Peak Rt (min) MS1 MS2 (key fragments) Tentative identification1 230 35308773 [M-H]minus 191055 179034 135045 neo-Chlorogenic acid2 322 35308745 [M-H]minus 191055 Chlorogenic acid3 354 35308773 [M-H]minus 191055 179034 173045 135044 crypto-Chlorogenic acid4 594 46308838 [M-H]minus 300027 Quercetin-galactoside5 605 46308804 [M-H]minus 300027 Quercetin-glucoside6 643 51511964 [M-H]minus 353087 335076 191055 179034 173045 135045 34-Dicaffeoylquinic acid7 667 51511970 [M-H]minus 353087 191055 179034173045 135044 35-Dicaffeoylquinic acid8 707 51511951 [M-H]minus 353087 191055 179034173045 135044 45-Dicaffeoylquinic acid9 728 53526876 [M+Cl]minus 337238 C26H44O9Cl10 754 33723821 [M-H]minus mdash C20H33O411 852 33723840 [M-H]minus mdash C20H33O4

Table 2 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 1st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 564 plusmn 070 037 plusmn 006 049 plusmn 006 046 plusmn 003 0036 plusmn 00100 796 plusmn 015 1016 plusmn 189

C+ 499 plusmn 027 036 plusmn 002 044 plusmn 003 042 plusmn 002 0046 plusmn 00058 791 plusmn 007 1019 plusmn 066

ESAH (30mgkg) 381 plusmn 046 106 plusmn 007ab 067 plusmn 006 122 plusmn 008ab 0033 plusmn 00068 782 plusmn 019 1014 plusmn 223

ESAH (100mgkg) 396 plusmn 036 092 plusmn 019ab 053 plusmn 007 108 plusmn 023ab 0034 plusmn 00041 781 plusmn 013 1019 plusmn 210

ESAH (300mgkg) 344 plusmn 026 080 plusmn 012ab 049 plusmn 007 100 plusmn 008ab 0036 plusmn 00068 743 plusmn 014 1018 plusmn 176

ENAL 472 plusmn 059 027 plusmn 005 055 plusmn 003 037 plusmn 006 0041 plusmn 00019 796 plusmn 015 1017 plusmn 097

HCTZ 761 plusmn 028a 099 plusmn 024ab 075 plusmn 017 118 plusmn 030ab 0030 plusmn 00069 780 plusmn 013 1015 plusmn 117

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

respectively whileHR levels were reduced to 165plusmn77 bpm Inaddition ESAH at doses of 100 and 300mg was not ableto change the SBP DBP MBP or HR of normotensive or2K1CplusOVT-ratsHowever as expected ENAL andHCTZwere able to significantly reduce SBP DBP andMBP levels inpositive controls or Sham-operated animals Values obtainedwith ENAL or HCTZ administration on the arterial pressurelevels were not statistically different from those observed aftertreatment with ESAH (30mgkg)

34 Prolonged Treatment with ESAH Restores Vascular Reac-tivity in MVBs from 2K1C OVT-Rats In MVBs from 2K1Cplus OVT-rats the administration of ACh or SNP was able toinduce a vasodilatory response approximately 35 lower thanthat in Sham-operated rats (Figures 3(a) and 3(b)) Similarlythe vasoconstrictor response of PHE was significantly higherin the 2K1C plus OVT-group when compared to Sham-operated animals (Figure 3(c)) On the other hand in animalsthat received ESAH (30mgkg) or ENAL (15mgkg) theeffects of ACh SNP or PHE were not different from thoseobserved in Sham-operated animals Treatments with HCTZ(25mgkg) or ESAH at doses of 100 and 300mgkg were notable to reverse the changes in vascular reactivity observed in2K1C plus OVT-animals (Figures 3(a)ndash3(c))

35 ESAH Reduces Oxidative and Nitrosative Stress withoutAffectingAldosterone Vasopressin Levels or SerumACEActiv-ity The effects of ESAH ENAL and HCTZ on TBARS NT

and nitrite levels are shown in Figures 4(a) 4(b) and4(c) Ovariectomy associatedwith renovascular hypertensionincreases TBARS andNT levels insim104 and 60 respectivelyTreatment with ESAH (30 100 and 300mgkg) or ENALreduced TBARS and NT levels to values close to those foundin Sham-operated animals

At baseline nitrite concentration was significantly lowerin positive control animals when compared with Sham-operated ones (63 plusmn 53 versus 94 plusmn 50 120583M) On the otherhand treatment with ESAH (at all doses) or ENAL increasedthe nitrite levels by sim50

On the other hand only ENAL was able to reducethe aldosterone levels and serum ACE activity (Figures4(a)ndash4(c)) without affecting serum vasopressin Treatmentwith ESAH (30 100 and 300mgkg) orHCTZdid not signifi-cantly change the aldosterone and vasopressin concentrationsor serum ACE activity (Figures 5(a)ndash5(c))

36 ESAH Induce Vasodilatory Response in MVBs fromNormotensive and 2K1C Plus OVT-Rats in Dependence onEndothelial NO and Prostaglandins The continuous perfu-sion of the MVBs (from normotensive or 2K1C plus OVT-rats) with phenylephrine resulted in a sustained increase inthe vascular perfusion pressure which was significantlyreduced by ESAH (0001 0003 and 001mg) (Figures 6(b)and 6(d))The characterization of a typical experiment shownin Figure 6(a) (normotensive) or Figure 6(c) (2K1C plusOVT-rats) reveals that when the highest ESAH dose was

6 Evidence-Based Complementary and Alternative Medicine

0102030405060708090

100

Rela

tive a

bund

ance

038 041095 188 230 255

322354 423 494 587

594

643

667

728754

761825

852

872 913 946 10381067

1077

10891178 1241 1279 1311 1372 1435

708

179035 HO HO

HOHO

HO

HO

HO

HO

HO

O

O

O

O

O

OO

O

O

O

O

O

O OOH OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

191056

191056

353087

353087

353087191056

191056

191056

191056

179035

179035

179035

179035

135045135045

173045

173045

335077 335077

353087 353087191056

135045135045 135045335077

353087353087

353087

515119 515119 515119

173045

173045 173045179035

179035 179035

191056

191056191056

0102030405060708090

100Re

lativ

e abu

ndan

ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150Time (min)

0102030405060708090

100

Rela

tive a

bund

ance

OminusOminus

Ominus

Ominus

350250 300200150mz

350250 300200150mz

350250 300200150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

(a)

(b) (c) (d)

(e) (f) (g)

Figure 1 Phytochemical analysis of ESAH obtained from A hispidum DC (a) Base peak chromatogram obtained by UPLC detected byHR-MS in the negative ionization and the fragmentation profile of monocaffeoylquinic acids tentatively identified as neo-chlorogenic acid(5-CQA) (b) chlorogenic acid (3-CQA) (c) and crypto-chlorogenic acid (4-CQA) (d) The main breakdown site for dicaffeoylquinic acids isdepicted in the 35 dicaffeoylquinic acid (35-di-CQA) and the fragmentation profile observed for peak 643min (e) 667min (f) and 707min(g)

used (001mg) the vasodilatory effects of ESAHhad responseintensity similar to that of acetylcholine

Previous infusion with L-NAME significantly reducedpart of the effect on theMVBs obtained with ESAHThe peakeffect of 0001 0003 and 001mg of ESAH was decreasedfrom 28 plusmn 4 54 plusmn 6 and 68 plusmn 7 to 11 plusmn 3 22 plusmn 9 and

26 plusmn 11 in normotensive and from 24 plusmn 3 50 plusmn 7 and62 plusmn 9 to 9 plusmn 2 18 plusmn 7 and 16 plusmn 6 in 2K1C plus OVT-rats respectively (Figures 7(a) and 7(d)) In a similar waythe vasodilatory effect of ESAH at doses of 0001 0003 and001mg was reduced to 20 plusmn 7 25 plusmn 8 and 27 plusmn 9 innormotensive and to 14 plusmn 3 21 plusmn 6 and 20 plusmn 7 in 2K1C

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 5

Table 1 Phytochemical composition of ESAH obtained from A hispidum DC

Peak Rt (min) MS1 MS2 (key fragments) Tentative identification1 230 35308773 [M-H]minus 191055 179034 135045 neo-Chlorogenic acid2 322 35308745 [M-H]minus 191055 Chlorogenic acid3 354 35308773 [M-H]minus 191055 179034 173045 135044 crypto-Chlorogenic acid4 594 46308838 [M-H]minus 300027 Quercetin-galactoside5 605 46308804 [M-H]minus 300027 Quercetin-glucoside6 643 51511964 [M-H]minus 353087 335076 191055 179034 173045 135045 34-Dicaffeoylquinic acid7 667 51511970 [M-H]minus 353087 191055 179034173045 135044 35-Dicaffeoylquinic acid8 707 51511951 [M-H]minus 353087 191055 179034173045 135044 45-Dicaffeoylquinic acid9 728 53526876 [M+Cl]minus 337238 C26H44O9Cl10 754 33723821 [M-H]minus mdash C20H33O411 852 33723840 [M-H]minus mdash C20H33O4

Table 2 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 1st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 564 plusmn 070 037 plusmn 006 049 plusmn 006 046 plusmn 003 0036 plusmn 00100 796 plusmn 015 1016 plusmn 189

C+ 499 plusmn 027 036 plusmn 002 044 plusmn 003 042 plusmn 002 0046 plusmn 00058 791 plusmn 007 1019 plusmn 066

ESAH (30mgkg) 381 plusmn 046 106 plusmn 007ab 067 plusmn 006 122 plusmn 008ab 0033 plusmn 00068 782 plusmn 019 1014 plusmn 223

ESAH (100mgkg) 396 plusmn 036 092 plusmn 019ab 053 plusmn 007 108 plusmn 023ab 0034 plusmn 00041 781 plusmn 013 1019 plusmn 210

ESAH (300mgkg) 344 plusmn 026 080 plusmn 012ab 049 plusmn 007 100 plusmn 008ab 0036 plusmn 00068 743 plusmn 014 1018 plusmn 176

ENAL 472 plusmn 059 027 plusmn 005 055 plusmn 003 037 plusmn 006 0041 plusmn 00019 796 plusmn 015 1017 plusmn 097

HCTZ 761 plusmn 028a 099 plusmn 024ab 075 plusmn 017 118 plusmn 030ab 0030 plusmn 00069 780 plusmn 013 1015 plusmn 117

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

respectively whileHR levels were reduced to 165plusmn77 bpm Inaddition ESAH at doses of 100 and 300mg was not ableto change the SBP DBP MBP or HR of normotensive or2K1CplusOVT-ratsHowever as expected ENAL andHCTZwere able to significantly reduce SBP DBP andMBP levels inpositive controls or Sham-operated animals Values obtainedwith ENAL or HCTZ administration on the arterial pressurelevels were not statistically different from those observed aftertreatment with ESAH (30mgkg)

34 Prolonged Treatment with ESAH Restores Vascular Reac-tivity in MVBs from 2K1C OVT-Rats In MVBs from 2K1Cplus OVT-rats the administration of ACh or SNP was able toinduce a vasodilatory response approximately 35 lower thanthat in Sham-operated rats (Figures 3(a) and 3(b)) Similarlythe vasoconstrictor response of PHE was significantly higherin the 2K1C plus OVT-group when compared to Sham-operated animals (Figure 3(c)) On the other hand in animalsthat received ESAH (30mgkg) or ENAL (15mgkg) theeffects of ACh SNP or PHE were not different from thoseobserved in Sham-operated animals Treatments with HCTZ(25mgkg) or ESAH at doses of 100 and 300mgkg were notable to reverse the changes in vascular reactivity observed in2K1C plus OVT-animals (Figures 3(a)ndash3(c))

35 ESAH Reduces Oxidative and Nitrosative Stress withoutAffectingAldosterone Vasopressin Levels or SerumACEActiv-ity The effects of ESAH ENAL and HCTZ on TBARS NT

and nitrite levels are shown in Figures 4(a) 4(b) and4(c) Ovariectomy associatedwith renovascular hypertensionincreases TBARS andNT levels insim104 and 60 respectivelyTreatment with ESAH (30 100 and 300mgkg) or ENALreduced TBARS and NT levels to values close to those foundin Sham-operated animals

At baseline nitrite concentration was significantly lowerin positive control animals when compared with Sham-operated ones (63 plusmn 53 versus 94 plusmn 50 120583M) On the otherhand treatment with ESAH (at all doses) or ENAL increasedthe nitrite levels by sim50

On the other hand only ENAL was able to reducethe aldosterone levels and serum ACE activity (Figures4(a)ndash4(c)) without affecting serum vasopressin Treatmentwith ESAH (30 100 and 300mgkg) orHCTZdid not signifi-cantly change the aldosterone and vasopressin concentrationsor serum ACE activity (Figures 5(a)ndash5(c))

36 ESAH Induce Vasodilatory Response in MVBs fromNormotensive and 2K1C Plus OVT-Rats in Dependence onEndothelial NO and Prostaglandins The continuous perfu-sion of the MVBs (from normotensive or 2K1C plus OVT-rats) with phenylephrine resulted in a sustained increase inthe vascular perfusion pressure which was significantlyreduced by ESAH (0001 0003 and 001mg) (Figures 6(b)and 6(d))The characterization of a typical experiment shownin Figure 6(a) (normotensive) or Figure 6(c) (2K1C plusOVT-rats) reveals that when the highest ESAH dose was

6 Evidence-Based Complementary and Alternative Medicine

0102030405060708090

100

Rela

tive a

bund

ance

038 041095 188 230 255

322354 423 494 587

594

643

667

728754

761825

852

872 913 946 10381067

1077

10891178 1241 1279 1311 1372 1435

708

179035 HO HO

HOHO

HO

HO

HO

HO

HO

O

O

O

O

O

OO

O

O

O

O

O

O OOH OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

191056

191056

353087

353087

353087191056

191056

191056

191056

179035

179035

179035

179035

135045135045

173045

173045

335077 335077

353087 353087191056

135045135045 135045335077

353087353087

353087

515119 515119 515119

173045

173045 173045179035

179035 179035

191056

191056191056

0102030405060708090

100Re

lativ

e abu

ndan

ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150Time (min)

0102030405060708090

100

Rela

tive a

bund

ance

OminusOminus

Ominus

Ominus

350250 300200150mz

350250 300200150mz

350250 300200150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

(a)

(b) (c) (d)

(e) (f) (g)

Figure 1 Phytochemical analysis of ESAH obtained from A hispidum DC (a) Base peak chromatogram obtained by UPLC detected byHR-MS in the negative ionization and the fragmentation profile of monocaffeoylquinic acids tentatively identified as neo-chlorogenic acid(5-CQA) (b) chlorogenic acid (3-CQA) (c) and crypto-chlorogenic acid (4-CQA) (d) The main breakdown site for dicaffeoylquinic acids isdepicted in the 35 dicaffeoylquinic acid (35-di-CQA) and the fragmentation profile observed for peak 643min (e) 667min (f) and 707min(g)

used (001mg) the vasodilatory effects of ESAHhad responseintensity similar to that of acetylcholine

Previous infusion with L-NAME significantly reducedpart of the effect on theMVBs obtained with ESAHThe peakeffect of 0001 0003 and 001mg of ESAH was decreasedfrom 28 plusmn 4 54 plusmn 6 and 68 plusmn 7 to 11 plusmn 3 22 plusmn 9 and

26 plusmn 11 in normotensive and from 24 plusmn 3 50 plusmn 7 and62 plusmn 9 to 9 plusmn 2 18 plusmn 7 and 16 plusmn 6 in 2K1C plus OVT-rats respectively (Figures 7(a) and 7(d)) In a similar waythe vasodilatory effect of ESAH at doses of 0001 0003 and001mg was reduced to 20 plusmn 7 25 plusmn 8 and 27 plusmn 9 innormotensive and to 14 plusmn 3 21 plusmn 6 and 20 plusmn 7 in 2K1C

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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OphthalmologyJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Evidence-Based Complementary and Alternative Medicine

0102030405060708090

100

Rela

tive a

bund

ance

038 041095 188 230 255

322354 423 494 587

594

643

667

728754

761825

852

872 913 946 10381067

1077

10891178 1241 1279 1311 1372 1435

708

179035 HO HO

HOHO

HO

HO

HO

HO

HO

O

O

O

O

O

OO

O

O

O

O

O

O OOH OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

191056

191056

353087

353087

353087191056

191056

191056

191056

179035

179035

179035

179035

135045135045

173045

173045

335077 335077

353087 353087191056

135045135045 135045335077

353087353087

353087

515119 515119 515119

173045

173045 173045179035

179035 179035

191056

191056191056

0102030405060708090

100Re

lativ

e abu

ndan

ce

1 2 3 4 5 6 7 8 9 10 11 12 13 14 150Time (min)

0102030405060708090

100

Rela

tive a

bund

ance

OminusOminus

Ominus

Ominus

350250 300200150mz

350250 300200150mz

350250 300200150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

200 250 300 350 400 450 500150mz

0102030405060708090

100

Rela

tive a

bund

ance

0102030405060708090

100

Rela

tive a

bund

ance

(a)

(b) (c) (d)

(e) (f) (g)

Figure 1 Phytochemical analysis of ESAH obtained from A hispidum DC (a) Base peak chromatogram obtained by UPLC detected byHR-MS in the negative ionization and the fragmentation profile of monocaffeoylquinic acids tentatively identified as neo-chlorogenic acid(5-CQA) (b) chlorogenic acid (3-CQA) (c) and crypto-chlorogenic acid (4-CQA) (d) The main breakdown site for dicaffeoylquinic acids isdepicted in the 35 dicaffeoylquinic acid (35-di-CQA) and the fragmentation profile observed for peak 643min (e) 667min (f) and 707min(g)

used (001mg) the vasodilatory effects of ESAHhad responseintensity similar to that of acetylcholine

Previous infusion with L-NAME significantly reducedpart of the effect on theMVBs obtained with ESAHThe peakeffect of 0001 0003 and 001mg of ESAH was decreasedfrom 28 plusmn 4 54 plusmn 6 and 68 plusmn 7 to 11 plusmn 3 22 plusmn 9 and

26 plusmn 11 in normotensive and from 24 plusmn 3 50 plusmn 7 and62 plusmn 9 to 9 plusmn 2 18 plusmn 7 and 16 plusmn 6 in 2K1C plus OVT-rats respectively (Figures 7(a) and 7(d)) In a similar waythe vasodilatory effect of ESAH at doses of 0001 0003 and001mg was reduced to 20 plusmn 7 25 plusmn 8 and 27 plusmn 9 innormotensive and to 14 plusmn 3 21 plusmn 6 and 20 plusmn 7 in 2K1C

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

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Disease Markers

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 7th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 916 plusmn 109 043 plusmn 004 040 plusmn 006 047 plusmn 004 0036 plusmn 00064 796 plusmn 014 1009 plusmn 070

C+ 556 plusmn 021 049 plusmn 006 064 plusmn 008 059 plusmn 007 0050 plusmn 00034 806 plusmn 007 1011 plusmn 118

ESAH (30mgkg) 472 plusmn 057 105 plusmn 008ab 044 plusmn 004 112 plusmn 007ab 0037 plusmn 00061 748 plusmn 016 1014 plusmn 180

ESAH (100mgkg) 485 plusmn 071 098 plusmn 018ab 073 plusmn 011 128 plusmn 025ab 0059 plusmn 00032 795 plusmn 018 1011 plusmn 187

ESAH (300mgkg) 487 plusmn 061 083 plusmn 011 049 plusmn 008 094 plusmn 013ab 0032 plusmn 00089 755 plusmn 012 1014 plusmn 182

ENAL 603 plusmn 046 037 plusmn 003 048 plusmn 004 053 plusmn 003 0009 plusmn 00018 805 plusmn 014 1009 plusmn 102

HCTZ 685 plusmn 047 060 plusmn 007 072 plusmn 005 079 plusmn 008 0013 plusmn 00048 785 plusmn 011 1011 plusmn 084

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

ESAH (mgkg)B

ABAB AB

0

100

200

SBP

(mm

Hg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

AB ABA

B

0

100

200

DBP

(mm

Hg)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

B

AB AB AB

0

100

200

MA

P (m

m H

g)

ESAH (mgkg)

(c)

Sham C (+) 30 100 300 ENAL HCTZ

AB

B

0

200

400

HR

(bpm

)

ESAH (mgkg)

(d)

Figure 2 Prolonged administration of ESAH obtained from A hispidum reduces SBP (a) DBP (b) MAP (c) and HR (d) in 2K1C plus OVT-rats Vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg) was given orally for 28 days The letter ldquoC+rdquo indicatesthe effect measured after administration of vehicle only The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performedby means of one-way analysis of variance (ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+)B119901 lt 005 when compared to Sham-operated group ENAL enalapril DBP diastolic blood pressure HCTZ hydrochlorothiazide HR heartrate MAP mean arterial pressure SBP systolic blood pressure Sham placebo surgery

plus OVT-rats in preparations perfused with indomethacin(Figures 7(b) and 7(e)) Interestingly simultaneous treatment(coadministration) with L-NAME and indomethacin (Fig-ures 7(c) and 7(f)) vanished the vasorelaxation effect inducedby all ESAH doses inMVBs from normotensive or 2K1C plusOVT-rats

4 Discussion

In the present study it was demonstrated that 28-day ESAHtreatment was effective in inducing an important salureticand antihypertensive response in 2K1C plus OVT-rats Thisfavorable effect induced by ESAH is associated with a parallel

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Table 4 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 14th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)Sham 1036 plusmn 132 053 plusmn 006 050 plusmn 006 060 plusmn 008 0050 plusmn 00056b 796 plusmn 016 1014 plusmn 070C+ 711 plusmn 062 036 plusmn 004 041 plusmn 003 042 plusmn 005 0052 plusmn 00049 760 plusmn 008 1014 plusmn 086ESAH (30mgkg) 566 plusmn 023 050 plusmn 004 068 plusmn 006 060 plusmn 004 0030 plusmn 00052 807 plusmn 005 1017 plusmn 089ESAH (100mgkg) 478 plusmn 064 088 plusmn 020 050 plusmn 006 101 plusmn 024 0036 plusmn 00083 761 plusmn 020 1018 plusmn 212

ESAH (300mgkg) 576 plusmn 057 116 plusmn 003ab 074 plusmn 007 133 plusmn 004ab 0036 plusmn 00069 795 plusmn 008 1019 plusmn 088ENAL 601 plusmn 073 053 plusmn 008 056 plusmn 003 071 plusmn 008 0026 plusmn 00046 804 plusmn 003 1015 plusmn 102HCTZ 655 plusmn 033 074 plusmn 011 065 plusmn 010 092 plusmn 015 0055 plusmn 00232 806 plusmn 003 1014 plusmn 115

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

3 10 30

minus100

minus50

0

ShamC (+)

A

ACh (pmol)

AAA

AA

A

A

AA

A

A

Dec

reas

e in

perf

usio

n pr

essu

re (

)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(a)

3 10 30

minus100

minus50

0

A

SNP (pmol)

A

AAA

A

A

AD

ecre

ase i

n pe

rfus

ion

pres

sure

() A

A A

ShamC (+)

HCTZ (25 mgkg)ENAL (15 mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(b)

3 10 300

90

180

A

PHE (nmol)

AA

AA

A

AAA

Incr

ease

in p

erfu

sion

pres

sure

()

A

AA

ShamC (+)

HCTZ (25 mgkg)ENAL (15mgkg)ESAH (300 mgkg)

ESAH (100 mgkg)ESAH (30 mgkg)

(c)

Figure 3 Prolonged treatment with ESAH restores vascular reactivity inMVBs from 2K1C plus OVT-rats Effects of ACh (3 10 and 30 pmol(a)) SNP (3 10 and 30 pmol (b)) or PHE (3 10 and 30 nmol (c)) on the perfusion pressure of theMVBs from normotensive (Sham) or 2K1Cplus OVT-rats in the presence or absence of prolonged treatment with ESAH (30 100 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)Values in panel are expressed as mean plusmn SEM of 5 experiments A indicates 119901 lt 005 compared with the perfusion pressure in Sham-operatedrats All experiments were performed in endothelium-intact preparations ACh acetylcholine ENAL enalapril HCTZ hydrochlorothiazideSNP sodium nitroprusside OVT ovariectomy PHE phenylephrine

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

Table 5 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 21st day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 898 plusmn 093 046 plusmn 006 048 plusmn 005 052 plusmn 007 0041 plusmn 00070 787 plusmn 012 1015 plusmn 272

C+ 716 plusmn 042 059 plusmn 007 076 plusmn 011 074 plusmn 010 0068 plusmn 00078 810 plusmn 008 1014 plusmn 111

ESAH (30mgkg) 673 plusmn 072 073 plusmn 012 072 plusmn 018 099 plusmn 017 0043 plusmn 00054 825 plusmn 017 1014 plusmn 201

ESAH (100mgkg) 482 plusmn 033 077 plusmn 016 075 plusmn 020 075 plusmn 019 0046 plusmn 00054 830 plusmn 009 1018 plusmn 085

ESAH (300mgkg) 520 plusmn 123 121 plusmn 020ab 074 plusmn 006 147 plusmn 021ab 0039 plusmn 00033 785 plusmn 007 1019 plusmn 170

ENAL 530 plusmn 025 030 plusmn 006 051 plusmn 005 047 plusmn 006 0038 plusmn 00091 802 plusmn 006 1017 plusmn 172

HCTZ 622 plusmn 048 045 plusmn 006 041 plusmn 002 055 plusmn 005 0019 plusmn 00058 788 plusmn 006 1014 plusmn 095

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

B

AA

ab

B

AAB

0

10

20

TBA

RS (m

mol

l)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

B

AA ab

BA A

000

002

004

ESAH (mgkg)

NT

(m

oll)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

90

180

B

A A

a B

A

ESAH (mgkg)

Nitr

ite (

M)

(c)

Figure 4 ESAH treatment reduces TBARS (a) and NT (b) levels and increases NO bioavailability (c)The serum samples were obtained after28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ (25mgkg)The results show the mean plusmn SEM(119899 = 6-7) Statistical analyses were performed bymeans of one-way analysis of variance (ANOVA) followed byDunnettrsquos test A119901 lt 005whencompared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operated group ENAL enalapril HCTZ hydrochlorothiazideNT nitrosamine Sham placebo surgery TBARS thiobarbituric acid reactive substances

reduction of oxidative and nitrosative stress biomarkers inaddition to a possible increase in NO bioavailability Addi-tionally an important NO and prostaglandin-dependentvasodilator effect was observed in mesenteric vascular bed afinding that indicates a potential mechanism for the cardio-vascular effects of ESAH

In the first stage of our study we decided to use ENALand HCTZ as standard cardioprotective drugs The choice of

HCTZ was based on the need to compare the diuretic andnatriuretic effects of ESAH with a first-line diuretic drug inthe treatment of hypertension [17] On the other hand as therenovascular hypertension model used in our study (2K1C)induces a sustained activation of the renin-angiotensin sys-tem (RAS) we chose another classic drug (ENAL) capable ofeffectively blocking activation and preventing the installationof renovascular hypertension In addition data indicated that

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

Table 6 Effects of oral administration of purified aqueous extract obtained fromA hispidumDC (ESAH) on urinary volume and electrolyteexcretion pH and density on 28th day of treatment

Group Urinary volume ElNa+ Elk+ ElClminus ElCa++ pH Density(ml100 g8 h) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g) (120583Eqmin100 g)

Sham 784 plusmn 055 044 plusmn 003 049 plusmn 003 053 plusmn 004 0034 plusmn 00020 802 plusmn 004 1016 plusmn 136

C+ 592 plusmn 032 028 plusmn 002 037 plusmn 007 034 plusmn 004 0027 plusmn 00047 800 plusmn 020 1018 plusmn 092

ESAH (30mgkg) 485 plusmn 041 052 plusmn 004 066 plusmn 007 058 plusmn 004 0037 plusmn 00135 841 plusmn 005 1015 plusmn 108

ESAH (100mgkg) 468 plusmn 063 029 plusmn 002 043 plusmn 004 040 plusmn 003 0027 plusmn 00069 834 plusmn 007 1017 plusmn 204

ESAH (300mgkg) 394 plusmn 049 082 plusmn 009ab 077 plusmn 005 114 plusmn 012ab 0042 plusmn 00060 801 plusmn 008 1019 plusmn 108

ENAL 513 plusmn 061 072 plusmn 013 067 plusmn 010 088 plusmn 013 0046 plusmn 00061 831 plusmn 005 1014 plusmn 164

HCTZ 689 plusmn 045 080 plusmn 009ab 062 plusmn 007 096 plusmn 011 0032 plusmn 00133 804 plusmn 005 1017 plusmn 062

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+ a119901 lt 005) or Sham-operated group (b119901 lt 005)using one-way ANOVA followed by Dunnettrsquos test El excreted load HCTZ hydrochlorothiazide ENAL enalapril

Sham C (+) 30 100 300 ENAL HCTZ

0

110

220

BB

A

B B B

Plas

ma A

CE ac

tivity

(m

mol

min

ml)

ESAH (mgkg)

(a)

Sham C (+) 30 100 300 ENAL HCTZ

0

160

320

B B

A ab

B B B

Ald

oste

rone

(pg

ml)

ESAH (mgkg)

(b)

Sham C (+) 30 100 300 ENAL HCTZ

0

8

16

a

Vaso

pres

sin (p

mol

l)

ESAH (mgkg)

(c)

Figure 5 Prolonged treatment with ESAH does not affect plasma ACE activity (a) or serum levels of aldosterone (b) and vasopressin (c)The serum samples were obtained after 28 days of treatment with vehicle ESAH (30 100 and 300mgkg) ENAL (15mgkg) or HCTZ(25mgkg) The results show the mean plusmn SEM (119899 = 6-7) Statistical analyses were performed by means of one-way analysis of variance(ANOVA) followed by Dunnettrsquos test A119901 lt 005 when compared to 2K1C plus OVT-rats (C+) B119901 lt 005 when compared to Sham-operatedgroup ACE angiotensin converting enzyme ENAL enalapril HCTZ hydrochlorothiazide Sham placebo surgery

ACE inhibitors including ENAL would exert cardioprotec-tive effects regardless of the hypotensive action [18] Currentfindings have indicated that ACE inhibitors may increaseNO bioavailability and reduce oxidative and nitrosative stressparameters during hypertension These effects provide anadditional cardioprotective response to the sustained anti-hypertensive effect [19] Therefore considering our findingsENAL was presented as a standard drug for comparison withthe cardioprotective potential of ESAH

Another point that deserves attention is the initiationof treatments with ESAH ENAL and HCTZ only 4 weeksafter the surgical procedure This option stems from theneed to submit animals to a previous period of sustainedactivation of RAS and estrogen deprivation a limiting stepin oxidativenitrosative imbalance and in the establishmentof hypertension In fact all 2K1C plus OVT-rats had a highdegree of oxidativenitrosative stress accompanied by in-creased vascular reactivity to PHE and a reduced vasodilatory

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

Table 7 Effects of oral administration of purified aqueous extract obtained from A hispidum DC (ESAH) on serum Na+ K+ urea andcreatinine on 28th day of treatment

Group Na+ (mmolL) K+ (mmolL) Urea (mgdL) Creatinine (mgdL)Sham 1360 plusmn 035 481 plusmn 021 5337 plusmn 137 037 plusmn 001

C+ 1191 plusmn 383 447 plusmn 020 5507 plusmn 473 036 plusmn 002

ESAH (30mgkg) 1317 plusmn 170 616 plusmn 050 6368 plusmn 358 044 plusmn 003

ESAH (100mgkg) 1312 plusmn 554 495 plusmn 047 5443 plusmn 290 034 plusmn 001

ESAH (300mgkg) 1194 plusmn 889 474 plusmn 041 4940 plusmn 208 036 plusmn 001

ENAL 1344 plusmn 136 521 plusmn 010 6390 plusmn 135 040 plusmn 001

HCTZ 1365 plusmn 063 485 plusmn 021 6215 plusmn 264 035 plusmn 003

Values are expressed as mean plusmn SEM of 6-7 rats in each group in comparison with the positive control (C+) or Sham-operated group using one-way ANOVAfollowed by Dunnettrsquos test HCTZ hydrochlorothiazide ENAL enalapril

30

60

90ACh (nmol)

1 001

Phe

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Per

fusio

n pr

essu

re

(mm

Hg)

Normotensive

(a)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)Normotensive

(b)

30

60

90ACh (nmol)

1 001

5 10 15 20 25 30 35

0001 0003ESAH (mg)

Phe

2K1C plus OVT

Per

fusio

n pr

essu

re

(mm

Hg)

14

(c)

C 00003 0001 0003 001

minus80

minus40

0

A

AB

AB

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVT

(d)

Figure 6 ESAH induces vasodilatory response in MVBs from normotensive and 2K1C plus OVT-rats Effects of ESAH (0001 0003and 001mg) on the perfusion pressure of the MVBs from normotensive (b) and 2K1C plus OVT-rats (d) perfused with physiologicalsaline solution containing 3-120583M phenylephrine (PHE) Trace recording of the MVBs pressure showing the effects of the administrationof acetylcholine and ESAH in normotensive (a) and 2K1C plus OVT-rats (c) Values in panel are expressed as mean plusmn SEM of 5 experimentsA indicates 119901 lt 005 compared with the perfusion pressure recorded before ESAH B indicates 119901 lt 005 compared with the previous doseAll experiments were performed in endothelium-intact preparations

response to ACh and SNP showing a significant changein endothelial and vascular smooth muscle function Inaddition we observed a significant increase in ACE activityand aldosterone levels which may have contributed directlyto the onset of hypertension Treatment with ESAH signif-icantly reduced TBARS and NT levels and increased nitriteconcentration (an indirect marker of NO bioavailability)

showing important antioxidant and antinitrosant propertiesIn addition ESAH treatment restored vascular reactivity toPHE ACh and SNP demonstrating its significant vasopro-tective effects in 2K1C plus OVT-rats In fact this responsemay play a central role in the cardiorenal effects of ESAHsince unlike ENAL ESAH did not affect ACE activity andaldosterone levels

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Evidence-Based Complementary and Alternative Medicine

0001 0003 001

minus100

minus50

0

Vehicle

A

AA

ESAH (mg)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

Normotensive

L-NAME (100 M)

(a)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

NormotensiveVehicleIndomethacin (1M)

(b)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

Normotensive

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(c)

0001 0003 001

minus100

minus50

0A

AA

ESAH (mg)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

2K1C plus OVTVehicleL-NAME (100 M)

(d)

Dec

reas

e in

perf

usio

n pr

essu

re (

)

0001 0003 001

minus100

minus50

0

A A

ESAH (mg)

2K1C plus OVTVehicleIndomethacin (1M)

(e)D

ecre

ase i

n pe

rfus

ion

pres

sure

()

0001 0003 001

minus100

minus50

0A A A

ESAH (mg)

2K1C plus OVT

L-NAME (100 M) +indomethacin (1 M)

Vehicle

(f)

Figure 7 Role of NO and prostaglandins on the vasorelaxant effect of ESAH in MVBs from normotensive and hypertensive rats (2K1Cplus OVT) Effects of ESAH (0001 0003 and 001mg) on endothelium-intact MVBs continuously perfused with L-NAME ((a) and (d))indomethacin ((b) and (e)) or coadministration of L-NAME and indomethacin ((c) and (f)) The results show the mean plusmn SEM of 5preparations per group A indicates 119901 lt 005 compared with the effects of ESAH on the respective vehicle group

Whenwe look closely at the renal effects of ESAH we willsee that while HCTZ showed an expected diuretic responsewith an increase inwater and electrolyte elimination after firstadministration followed by a reduction of the diuretic effectby compensatory mechanisms ESAH did not affect the urinevolume eliminatedOn the other hand an important salureticeffect was evident especially on the elimination of Na+ andClminus throughout the experimental period In fact at the lowestdoses (30 and 100mgkg) the saluretic effect was evident onlyon the 1st and 7th days of treatment while renal Na+ and Clminuselimination continued significantly only at the highest dosetested (300mgkg) If we look at the antihypertensive effect ofESAHwewill see that themost significant reduction in bloodpressure levels also occurred at the lowest dose usedThis factmay explain in part the reduction of the saluretic responseobserved after the 7th day of treatment since an eventualreduction of renal hydrostatic pressure due to the sustainedreduction of blood pressure can reduce glomerular filtrationrate and renal salt and water elimination This effect wasalso evident with HCTZ a classic antihypertensive drug thatdespite inducing significant diuretic effect in the first doseshowed an important reduction in its efficacy after prolongedtreatment

It is known that cardiorenal regulation may involve sev-eral endogenous mediators including prostaglandins NOand the endothelium-derived hyperpolarizing factor syn-ergistically acting in a complex hemodynamic and neuro-humoral interaction [20] In our study it has been shownthat the cardiorenal effects induced by ESAH appear to beinfluenced by an expressive antioxidant activity which couldinfluence the increase in the bioavailability of NO andconsequently the vascular reactivity Although suggestivethere is still some doubt whether the effect is due to directrelease of NO by secondary metabolites present in ESAHor a consequence of the potent antioxidant activity The factis that substances that reduce reactive oxygen species mayincrease the bioavailability of NO and prostaglandins andconsequently induce systemic vasodilator responses [21]

In order to prove this hypothesis we evaluated the abilityof ESAH to reduce perfusion pressure in the MVBs fromnormotensive and 2K1C plus OVT-rats in the absence andpresence of L-NAME and indomethacin two drugs capableof inhibiting NO and prostaglandin synthase respectivelyThe putative blockade of NO and prostaglandin synthesisprevented the reduction of perfusion pressure induced byESAH in the MVBs suggesting that the release of NO and

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 13

prostaglandins could be involved in this effect A data thatreinforces our findings was described in a recent study [5]where it shows that an extract with a similar phytochemicalprofile was able to induce a NO-dependent acute hypotensiveresponse in normotensive rats evidencing the importanceof NO in the cardiovascular effects of ESAH Althoughwe believe that the cardiorenal effects presented in thisstudy result from a complex interaction among differentsecondary metabolites present in the ESAH the fact thattheremay be one ormore agents involved cannot be ruled outBiomonitoring studies can help answer these questions andif possible identify the agent that stands out in this process

5 Conclusions

A 28-day ESAH treatment reduces the blood pressure levelsin 2K1C-ovariectomized ratsThese effects are associatedwithan important antioxidant and antinitrosant action in addi-tion to a significant saluretic effect Probably this responsehas a direct contribution of the reduction in vascular re-sistance possibly mediated by the release of NO and pros-taglandins

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

This work was supported by grants from the Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPESBrazil) Fundacao de Apoio ao Desenvolvimento do EnsinoCiencia e Tecnologia do Estado deMatoGrosso do Sul (FUN-DECT Brazil 593000462015) and Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico (CNPq Brazil4494642014-8) The authors are grateful to Department ofBiochemistry and Molecular Biology of Federal Universityof Parana and University Hospital of the Federal Universityof Grande Dourados for mass spectrometry and biochemicalanalysis respectively

References

[1] A Sarma and N S Scott ldquoAssessing and modifying coronaryartery disease risk in womenrdquo Current Treatment Options inCardiovascular Medicine vol 19 no 7 2017

[2] R Lima M Wofford and J F Reckelhoff ldquoHypertension inpostmenopausal womenrdquoCurrentHypertension Reports vol 14no 3 pp 254ndash260 2012

[3] M L Muiesan M Salvetti C A Rosei and A Paini ldquoGenderDifferences inAntihypertensive TreatmentMyths or LegendsrdquoHigh Blood Pressure and Cardiovascular Prevention vol 23 no2 pp 105ndash113 2016

[4] P Rufus N Mohamed and A N Shuid ldquoBeneficial effects oftraditional Chinese medicine on the treatment of osteoporosison ovariectomised ratmodelsrdquoCurrent Drug Targets vol 14 no14 pp 1689ndash1693 2013

[5] C A Tirloni F A Lıvero R A Palozi et al ldquoEthnopharmaco-logical investigations of the cardio-renal properties of a nativespecies from the region of Pantanal state of Mato Grosso doSul Brazilrdquo Journal of Ethnopharmacology vol 206 pp 125ndash1342017

[6] I G C Bieski F Rios Santos R M De Oliveira et al ldquoEthno-pharmacology of medicinal plants of the pantanal region (MatoGrosso Brazil)rdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2012 Article ID 272749 36 pages 2012

[7] A Umar G Imam W Yimin et al ldquoAntihypertensive effects ofOcimum basilicum L (OBL) on blood pressure in renovascularhypertensive ratsrdquoHypertension Research vol 33 no 7 pp 727ndash730 2010

[8] S T Kau J R Keddie andDAndrews ldquoAmethod for screeningdiuretic agents in the ratrdquo Journal of Pharmacological Methodsvol 11 no 1 pp 67ndash75 1984

[9] A Gasparotto Jr M A Boffo E L B Lourenco M E AStefanello C A L Kassuya andM C A Marques ldquoNatriureticand diuretic effects of Tropaeolum majus (Tropaeolaceae) inratsrdquo Journal of Ethnopharmacology vol 122 no 3 pp 517ndash5222009

[10] D D McGregor ldquoThe effect of sympathetic nerve stimulationon vasoconstrictor responses in perfused mesenteric bloodvessels of the ratrdquo The Journal of Physiology vol 177 no 1 pp21ndash30 1965

[11] H H H W Schmidt P Wilke B Evers and E BohmeldquoEnzymatic formation of nitrogen oxides from L-arginine inbovine brain cytosolrdquo Biochemical and Biophysical ResearchCommunications vol 165 no 1 pp 284ndash291 1989

[12] R A S Santos E M Krieger and L J Greene ldquoAn improvedfluorometric assay of rat serum and plasma converting enzymerdquoHypertension vol 7 no 2 pp 244ndash252 1985

[13] N Dartora L M De Souza A P Santana-Filho et al ldquoUPLC-PDA-MS evaluation of bioactive compounds from leaves of Ilexparaguariensiswith different growth conditions treatments andageingrdquo Food Chemistry vol 129 no 4 pp 1453ndash1461 2011

[14] J Carlotto L M Da Silva N Dartora et al ldquoIdentification of adicaffeoylquinic acid isomer from Arctium lappa with a potentanti-ulcer activityrdquo Talanta vol 135 pp 50ndash57 2015

[15] MCarini RMaffei FacinoGAldiniMCalloni andLColomboldquoCharacterization of phenolic antioxidants from Mate (Ilexparaguayensis) by liquid chromatographymass spectrometryand liquid chromatographytandem mass spectrometryrdquo RapidCommunications in Mass Spectrometry vol 12 no 22 pp 1813ndash1819 1998

[16] LM de SouzaNDartora C T Scoparo P A J GorinM Iaco-mini and G L Sassaki ldquoDifferentiation of flavonol glucosideand galactoside isomers combining chemical isopropylidena-tion with liquid chromatography-mass spectrometry analysisrdquoJournal of Chromatography A vol 1447 pp 64ndash71 2016

[17] D Cooney S Milfred-LaForest andM Rahman ldquoDiuretics forhypertension Hydrochlorothiazide or chlorthalidonerdquo Cleve-land Clinic journal of medicine vol 82 no 8 pp 527ndash533 2015

[18] P P Toth ldquoPleiotropic effects of angiotensin receptor blockersaddressing comorbidities by optimizing hypertension therapyrdquoJournal of Clinical Hypertension vol 13 no 1 pp 42ndash51 2011

[19] A Wzgarda R Kleszcz M Prokop et al ldquoUnknown face ofknown drugs ndash what else can we expect from angiotensin con-verting enzyme inhibitorsrdquo European Journal of Pharmacologyvol 797 pp 9ndash19 2017

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Evidence-Based Complementary and Alternative Medicine

[20] M Feletou and P M Vanhoutte ldquoEndothelium-derived hyper-polarizing factor where are we nowrdquo Arteriosclerosis Throm-bosis and Vascular Biology vol 26 no 6 pp 1215ndash1225 2006

[21] P Zizkova M Stefek L Rackova M Prnova and L HorakovaldquoNovel quercetin derivatives from redox properties to promis-ing treatment of oxidative stress related diseasesrdquo Chemico-Biological Interactions vol 265 pp 36ndash46 2017

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom