Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2017 / Article

Research Article | Open Access

Volume 2017 |Article ID 2492483 | 14 pages | https://doi.org/10.1155/2017/2492483

Roles of Nitric Oxide and Prostaglandins in the Sustained Antihypertensive Effects of Acanthospermum hispidum DC. on Ovariectomized Rats with Renovascular Hypertension

Academic Editor: Jian-Li Gao
Received28 Jun 2017
Revised10 Aug 2017
Accepted16 Aug 2017
Published20 Sep 2017

Abstract

Although Acanthospermum hispidum is used in Brazilian folk medicine as an antihypertensive, no study evaluated its effects on a renovascular hypertension and ovariectomy model. So, this study investigated the mechanisms involved in the antihypertensive effects of an ethanol-soluble fraction obtained from A. hispidum (ESAH) using two-kidney-one-clip hypertension in ovariectomized rats (2K1C plus OVT). ESAH was orally administered at doses of 30, 100, and 300 mg/kg, daily, for 28 days, after 5 weeks of surgery. Enalapril (15 mg/kg) and hydrochlorothiazide (25 mg/kg) were used as standard drugs. Diuretic activity was evaluated on days 1, 7, 14, 21, and 28. Systolic, diastolic, and mean blood pressure and heart rate were recorded. Serum creatinine, urea, thiobarbituric acid reactive substances, nitrosamine, nitrite, aldosterone, vasopressin levels, and ACE activity were measured. The vascular reactivity and the role of nitric oxide (NO) and prostaglandins (PG) in the vasodilator response of ESAH on the mesenteric vascular bed (MVB) were also investigated. ESAH treatment induced an important saluretic and antihypertensive response, therefore recovering vascular reactivity in 2K1C plus OVT-rats. This effect was associated with a reduction of oxidative and nitrosative stress with a possible increase in the NO bioavailability. Additionally, a NO and PG-dependent vasodilator effect was observed on the MEV.

1. Introduction

In recent years, evidence has shown that there are significant differences between the genesis and the development of cardiovascular diseases between men and women [1]. In general, women are more affected by cardiovascular diseases, especially hypertension, after a pronounced drop in estrogen levels, a fact that usually occurs after menopause. It is now known that one of the mechanisms by which blood pressure may be elevated in aging postmenopausal women is the activation of the renin-angiotensin system (RAS). Postmenopausal women exhibit an increase in plasma renin activity, suggesting activation of the RAS similarly to what occurs during renovascular hypertension [2]. Approximately 50% of menopausal women have hypertension, a condition that associated with other estrogen-related risk factors (such as obesity and dyslipidemia) significantly contributes to acute cardiovascular events [3]. In contrast, although this condition is quite common, preclinical studies specifically designed for this type of population are still very restricted [4].

Recently, we have shown that the ethanol-soluble fraction obtained from Acanthospermum hispidum DC. (Asteraceae) (ESAH), an important medicinal species widely used in Brazil, has a significant acute hypotensive effect on normotensive male Wistar rats [5]. In addition, we have also shown that ESAH, rich in phenolic compounds, such as derivatives of caffeic acid and glycosylated flavonoids (quercetin glucoside and galactoside), does not produce any toxic effects after acute treatment [5]. So, despite the widespread use of A. hispidum as an antihypertensive by the Brazilian population [6], its prolonged diuretic and antihypertensive effects have not yet been scientifically evaluated.

Thus, in this work, the prolonged diuretic and antihypertensive effects of A. hispidum on ovariectomized rats with renovascular hypertension were investigated to simulate a broad part of the woman population aged over 50 years. In addition, the molecular mechanisms involved in A. hispidum antihypertensive response using isolated mesenteric vascular bed (MVB) were also evaluated.

2. Materials and Methods

2.1. Drugs

The following drugs, salts, and solutions were used: xylazine and ketamine hydrochloride (from Syntec, São Paulo, SP, Brazil), heparin (from Hipolabor, Belo Horizonte, MG, Brazil), and acetylcholine chloride, phenylephrine, indomethacin, Nω-nitro-L-arginine methyl ester (L-NAME), sodium nitroprusside, NaCl, KCl, NaHCO3, MgSO4, CaCl2, KH2PO4, dextrose, and ethylenediaminetetraacetic acid (EDTA) (all purchased from Sigma-Aldrich, Saint Louis, MO, USA).

2.2. Phytochemical Study
2.2.1. Plant Material and Preparation of the Purified Aqueous Extract

Aerial parts of Acanthospermum hispidum were collected from the botanical garden of the Federal University of Grande Dourados (UFGD, Dourados, Brazil) at 458 m above sea level (S 22°11′42.7′′ and W 54°56′10.2′′), in October 2015. A voucher specimen was authenticated by Dra. Maria do Carmo Vieira (DDMS number 5219) and deposited at the UFGD plant facility.

A. hispidum aqueous extract was obtained by infusion in a similar manner to that popularly used in Brazil [6] and prepared according to Tirloni et al. [5]. For this, A. hispidum leaves were air-dried in an oven at 40°C for 7 days and then the dried plant was cut and ground into powder form using mechanical milling. The extract was obtained by infusing 1 liter of boiling water for each 60 grams of dried and pulverized plant. Extraction was carried out until room temperature was reached (~5 h). The infusion was treated with 3 volumes of EtOH, which gave rise to a precipitate and an ethanol-soluble fraction (ESAH). ESAH was filtered, ethanol was totally removed by evaporation, and the resulting fraction was lyophilized (yield of 8.05%). All preparations were kept in freezer until further analyses.

2.2.2. Sample Analysis (Liquid Chromatography-Mass Spectrometry (LC-MS))

Chromatography was performed in an ultra-performance liquid chromatography (UPLC™ Waters), using a reversed phase column HSS T3 C18 column, with 100 × 2.1 mm and 1.7 μm of particle size (Waters), with constant temperature of 60°C. The solvents were ultra-pure water (Milli-Q, Millipore) and acetonitrile (JT Baker) containing 0.1% (v/v) of formic acid 96% (Tedia), and the gradient was performed increasing the acetonitrile from 0 to 10%, in 6 min, then to 80% in 14 min, at flow rate of 0.4 ml/min. The solvent returned to the initial condition in 15 min and the system was reequilibrated for 2 min. The sample was prepared at 2 mg/mL in MeOH-H2O (1 : 1, v/v), with injections of 5 μL.

The detection was provided by a high-resolution mass spectrometer, LTQ Orbitrap XL (Thermo Scientific). The ions were detected in the negative and positive modes. The ion source was held at 350°C and the desolvation was aided by nitrogen stream, at 40 arbitrary units in the sheath gas and 10 a.u. in the auxiliary gas. Negative ions provided best results for the sample, with the energies at 3.5 kV in the source, −46 V in the capillary, and −200 V in the tube lens. Fragmentations were performed by higher-energy collisional dissociation, with normalized collision energy of 25–35. For LC-MS analysis, the MS resolution was set at 15,000 FWHM mass accuracy which was obtained by external calibration, routinely performed.

2.3. Pharmacological Studies
2.3.1. Animals

Twelve-week-old female Wistar rats weighing 200–300 g were randomized and housed in plastic cages, with environmental enrichment, at °C under a 12/12 h light dark cycle, % humidity conditions, with ad libitum access to food and water. All procedures were approved by the ethical committee on animal use of the Federal University of Grande Dourados (UFGD) (number 45/2016), and experiments were performed in accordance with international standards and ethical guidelines on animal welfare.

2.3.2. Ovariectomy and Induction of Renovascular Hypertension

Rats were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) by intraperitoneal route. After ovariectomy (OVT), renovascular hypertension was induced using the Goldblatt model (two kidneys, one clip; 2K1C) as described by Umar et al. [7]. The left renal artery was exposed by retroperitoneal incision and dissected. A silver clip (lumen of 0.22 mm) was placed around the artery for partial occlusion. In the Sham-operated group (placebo surgery), the artery was not clipped and ovaries were not removed. After surgery, animals received saline for rehydration (2 ml/animal, subcutaneously, single administration), anti-inflammatory (indomethacin, 2 mg/kg, by oral route, every 12 hours, during 3 days), and antibiotic (enrofloxacin, 10 mg/kg, subcutaneously, single dose). Systolic blood pressure (SBP) was weekly measured (for 4 weeks) using the tail-cuff method. Only hypertensive rats (SBP above 140 mm Hg) were used in experiments.

2.3.3. Experimental Groups

Five weeks after surgery, animals were randomized and divided into 7 groups (-7) for hemodynamic and renal function studies. Rats were treated once a day (by gavage), during 28 days, with vehicle (2K1C plus OVT = positive control group), hydrochlorothiazide (HCTZ, 25 mg/kg, standard diuretic drug), enalapril (ENAL, 15 mg/kg, standard antihypertensive drug), or ESAH (30, 100, or 300 mg/kg). The Sham-operated group received vehicle under the same conditions.

2.3.4. Diuretic Activity

The diuretic effects of ESAH were accessed according to methods previously described by Kau et al. [8] with some modifications [9]. Weekly (on days 1, 7, 14, 21, and 28), rats were fasted overnight (6 h) with free access to water. Animals received an oral load of isotonic saline (0.9% NaCl, 5 ml/100 g) to impose a controlled water and salt balance before treatments. Then, rats were immediately placed in metabolic cages. Urine samples were collected in a graduated cylinder and the volume was recorded 8 hours after treatment (expressed as ml per 100 g body weight). During the experiment, rats were food deprived. pH and density were determined on fresh urine samples using digital pH meter (Q400MT; Quimis Instruments, Brazil) and a handheld refractometer (NO107; Nova Instruments, Brazil), respectively. Urinary sodium (Na+), potassium (K+), chloride (Cl-), and calcium (Ca++) levels were quantified in automated analyzer (Roche® Cobas Integra 400 plus). Excretion load (El) of Na+, K+, Cl-, and Ca++ was obtained according to the equation , where is the concentration of electrolytes (mEq/L) and is the urinary flow (mL/min). Results were expressed as μEq/min/100 g.

2.3.5. Blood Pressure and Heart Rate Evaluation

At the end of the trial period (day 28), rats were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) by intramuscular route. Immediately, a bolus injection of heparin (50 IU) was subcutaneously applied. The left carotid artery was cannulated and connected to a pressure transducer coupled to a PowerLab® recording system, and an application software (Chart, v 4.1; all from ADI Instruments, Castle Hill, Australia), recording systolic blood pressure (SBP) and diastolic blood pressure (DBP), mean blood pressure (MBP), and heart rate (HR). After 15 minutes of stabilization, changes in blood pressure and HR were recorded for 5 min.

2.3.6. Mesenteric Vascular Reactivity

After systemic blood pressure and HR measurements, and before euthanasia, the mesenteric vascular bed (MVB) was isolated and prepared for perfusion as previously described [10]. The isolated MVBs were then placed in a water-jacketed organ bath maintained at 37°C and perfused with PSS (composition in mM: NaCl 119; KCl 4.7; CaCl2 2.4; MgSO4 1.2; NaHCO3 25.0; KH2PO4 1.2; dextrose 11.1; and EDTA 0.03) gassed with 95% O2/5% CO2 at constant flow rate of 4 ml/min through a peristaltic pump. Changes in the perfusion pressure (mm Hg) were measured by a pressure transducer connected to an acquisition system (PowerLab) and its application software (Chart, v 7.1; both from ADI Instruments, Castle Hill, Australia). After setup in the perfusion apparatus, preparation was allowed to equilibrate for 30 to 45 min, and its viability was checked by a bolus injection of KCl (120 mmol). After a 30-minute stabilization period, a phenylephrine (PHE; 3, 10, and 30 nmol) dose-response administration was performed by injecting increasing doses into the perfusion system. After a 60-minute equilibration period, MVBs were continuously perfused with PSS containing 3 μM PHE, which was enough to induce a sustained increased perfusion pressure. Under these conditions, vasodilatory effects of acetylcholine (ACh; 3, 10, and 30 pmol) or sodium nitroprusside (SNP; 3, 10, and 30 pmol) were measured. A 15-minute equilibration period was allowed between each drug. All drugs were given into the perfusate as bolus injection of 100 μl.

2.3.7. Serum Biochemical Parameters

At the end of experiments, all animals were killed by suprapharmacological isoflurane dose and blood samples were collected. Serum was obtained by centrifugation (1,000 ×g for 10 min). Serum Na+, K+, urea, and creatinine were determined in automated biochemical analyzer (Roche Cobas Integra 400 plus). Serum nitrotyrosine (NT), aldosterone, and vasopressin levels were measured by enzyme-linked immunosorbent assay kit according to manufacturer’s specifications (BD Biosciences, CA, USA). Thiobarbituric acid (TBARS) levels were measured using TBARS assay kits (Cayman Chemical, Ann Arbor, Michigan, USA) according to manufacturer’s instruction. Plasma nitrite concentration was determined by enzymatically reducing nitrate according to technique described by Schmidt et al. [11]. Serum angiotensin converting enzyme (ACE) activity was determined by indirect fluorimetry according to methods described by Santos et al. [12].

2.4. Investigation of the Molecular Mechanisms Involved in the Vasodilatory Response of ESAH
2.4.1. Evaluation of the Effects of ESAH on the Mesenteric Vascular Bed

Preparations (MVBs) with functional endothelium from normotensive and hypertensive (2K1C plus OVT) female rats, without any previous treatment, were continuously perfused with PSS containing PHE (3 μM). After stabilization of the increased perfusion pressure, preparations received bolus injections containing 0.0003, 0.001, 0.003, and 0.01 mg of ESAH, and the reduction in the perfusion pressure was evaluated. Each next dose was administered only after the return of the perfusion pressure to the same level recorded before injection, with minimal interval of 3 min between doses.

2.4.2. Investigation of the Mechanisms Involved in the Vascular Effects of ESAH

In these experiments, after recording the first dose-response curve to ESAH (0.001, 0.003, and 0.01 mg), the MVBs were left to equilibrate for an additional period of 30–45 min under perfusion with PSS. Then, different preparations were perfused with PSS containing 3 μM phenylephrine plus indomethacin (1 μM; a nonselective cyclooxygenase inhibitor) and L-NAME (100 μM; a nonselective NO synthase inhibitor) alone or combined. After 15 min under perfusion with one of the previously mentioned solutions, ESAH (0.001, 0.003, and 0.01 mg) was injected again into the perfusion system. The ability of ESAH to reduce the perfusion pressure was compared with results obtained in control preparations, perfused with vehicle only.

2.5. Statistical Analyses

Data were analyzed for homogeneity of variance and normal distribution. Differences between means were determined using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The level of significance was set at 95% (), and results are expressed as mean ± standard error of the mean (SEM). Graphs were drawn and statistical analysis was carried out using GraphPad® Prism software version 5.0 for Mac OS X (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Phytochemical Characterization

In a previous work [6], the composition of an extract of Acanthospermum hispidum was partially characterized, with identification of the family of monocaffeoylquinic acids (CQAs) and dicaffeoylquinic acids (diCQAs), observed as multiple isomers in the LC-MS analysis. However, these isomers have characteristic fragmentation profile that can be used for their identification [13, 14]. To confirm the isomer identity, an extract of Ilex paraguariensis, containing mono- and dicaffeoylquinic acids, previously characterized [13], was used as a reference sample.

The detailed chemical composition of ESAH obtained from A. hispidum is shown in Table 1. The negative ionization was better for the acidic compounds present in the extract, and the monocaffeoylquinic acids appeared at 353.087. The carbons from the ring of quinic acid were numerated according to the chlorogenic acid structure, considered as 3-monocaffeoylquinic acid (3-CQA). Thus, based on the fragmentation profile (at collision energy of 25) and retention time, compared to I. paraguariensis extract, the isomers could be predicted as neo-chlorogenic acid (5-CQA at 2.30 min), with intense fragments at 191.056, 179.035, and 135.045 but absent of 173.45. The chlorogenic acid (3-CQA) appeared at 3.22 min, with a main fragment at 191.056 and the crypto-chlorogenic acid (4-CQA at 3.54 min) produced fragments at 191.056, 179.035, 173.045, and 135.045, detaching the most abundant fragment-ion at 173.045 (Figure 1). These results are in accordance with the pioneer work of Carini et al. [15]. The peaks at 5.94 and 6.05 min were observed at 463.088, with similar fragments at 300.027, 271.024, 255.029, and 243.029, consistent with quercetin-hexosides, being consistent with quercetin galactoside and quercetin glucoside [16].


PeakRt (min)MS1MS2 (key fragments)Tentative identification

12.30353.08773 [M-H]191.055, 179.034, 135.045neo-Chlorogenic acid
23.22353.08745 [M-H]191.055Chlorogenic acid
33.54353.08773 [M-H]191.055, 179.034, 173.045, 135.044crypto-Chlorogenic acid
45.94463.08838 [M-H]300.027Quercetin-galactoside
56.05463.08804 [M-H]300.027Quercetin-glucoside
66.43515.11964 [M-H]353.087, 335.076, 191.055, 179.034, 173.045, 135.0453,4-Dicaffeoylquinic acid
76.67515.11970 [M-H]353.087, 191.055, 179.034,173.045, 135.0443,5-Dicaffeoylquinic acid
87.07515.11951 [M-H]353.087, 191.055, 179.034,173.045, 135.0444,5-Dicaffeoylquinic acid
97.28535.26876 [M+Cl]337.238C26H44O9Cl
107.54337.23821 [M-H]C20H33O4
118.52337.23840 [M-H]C20H33O4

The main peaks on chromatogram were identified as dicaffeoylquinic acids (di-CQAs) at 515.119, and similar to the CQAs, they differ in their fragmentation profile obtained with collision energy at 35. The peak at 6.43 min was consistent with 3,4-di-CQA, with fragments at 353.087 due to the loss of a caffeoyl residue, and the fragment at 335.076 (353.087, H2O) was observed at a relative intensity in only this isomer. The other fragments were 191.056 (quinic acid, base peak), 179.035 (caffeic acid), and 173.045 (quinic acid, H2O). The peaks at 6.67 and 7.07 min presented a quite similar fragmentation profile, containing almost all fragments observed for the peak at 6.43 min, except by the absence of ion at 335.076. The chromatographic profile suggests the peak at 6.67 min as 3,5-di-CQA and the peak at 7.07 min as 4,5-di-CQA. However, other isomers may be eluted in very close retention time as previously observed [14], hindering the accurate isomer identification. Other peaks (7.28, 7.54, and 8.52 min) showed the main ion at 337.238, which could be result of in-source fragmentation and remains unidentified and the peaks at 10.38, 10.67, 10.77, and 10.89 min presented multiple ions avoiding their identification.

3.2. Prolonged Treatment with ESAH Induced Significant Saluretic Effect on Sham-Operated and 2K1C Plus OVT-Rats

The values obtained for urinary volume and renal excretion of electrolytes are shown in Tables 2, 3, 4, 5, and 6. The mean urinary volumes of Sham-operated rats collected for 8 hours on days 1, 7, 14, 21, and 28 were, respectively, , , , , and  ml/100 g/8 h. Similarly, 2K1C plus OVT-animals produced an estimated urinary volume of , , , , and  ml/100 g/8 h on days 1, 7, 14, 21, and 28, respectively. None of the groups treated with ENAL or ESAH (30, 100, and 300 mg/kg) had any changes in urinary volume when compared to Sham-operated or 2K1C plus OVT-rats. As expected, animals that received HCTZ had a significant increase in urinary volume only on the 1st day of treatment.


GroupUrinary volume pHDensity
(ml/100 g/8 h)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL
HCTZ

Values are expressed as mean ± SEM of 6-7 rats in each group in comparison with the positive control (C+; ) or Sham-operated group () using one-way ANOVA followed by Dunnett’s test. El: excreted load; HCTZ: hydrochlorothiazide; ENAL: enalapril.

GroupUrinary volumepHDensity
(ml/100 g/8 h)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL
HCTZ

Values are expressed as mean ± SEM of 6-7 rats in each group in comparison with the positive control (C+; ) or Sham-operated group () using one-way ANOVA followed by Dunnett’s test. El: excreted load; HCTZ: hydrochlorothiazide; ENAL: enalapril.

GroupUrinary volume pHDensity
(ml/100 g/8 h)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL
HCTZ

Values are expressed as mean ± SEM of 6-7 rats in each group in comparison with the positive control (C+; ) or Sham-operated group () using one-way ANOVA followed by Dunnett’s test. El: excreted load; HCTZ: hydrochlorothiazide; ENAL: enalapril.

GroupUrinary volume pHDensity
(ml/100 g/8 h)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL
HCTZ

Values are expressed as mean ± SEM of 6-7 rats in each group in comparison with the positive control (C+; ) or Sham-operated group () using one-way ANOVA followed by Dunnett’s test. El: excreted load; HCTZ: hydrochlorothiazide; ENAL: enalapril.

GroupUrinary volume pHDensity
(ml/100 g/8 h)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)(µEq/min/100 g)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL
HCTZ

Values are expressed as mean ± SEM of 6-7 rats in each group in comparison with the positive control (C+; ) or Sham-operated group () using one-way ANOVA followed by Dunnett’s test. El: excreted load; HCTZ: hydrochlorothiazide; ENAL: enalapril.

On the 1st day of treatment, all animals treated with ESAH (30, 100, or 300 mg/kg) or HCTZ presented a significant increase in Na+ and Cl- elimination, with saluretic index higher than 2.0 (Table 2). On the other hand, only animals treated with ESAH (30, 100, and 300 mg/kg) maintained this effect on the 7th day of treatment (Table 3). On the 14th, 21st, and 28th days of treatment, only animals receiving ESAH at the dose of 300 mg/kg had significant urinary Na+ and Cl- levels (Tables 4, 5, and 6). In contrast, animals treated with HCTZ returned to significant Na+ levels only on the 28th day of treatment (Table 6). Groups receiving ENAL did not show any significant increase in urinary Na+, K+, Cl-, or Ca++ when compared with positive controls or Sham-operated animals. Similarly, urinary pH and density or serum Na+, K+, urea, and creatinine values were not altered by any of the treatments (Tables 2, 3, 4, 5, 6, and 7).


GroupNa+ (mmol/L)K+ (mmol/L)Urea (mg/dL)Creatinine (mg/dL)

Sham
C+
ESAH (30 mg/kg)
ESAH (100 mg/kg)
ESAH (300 mg/kg)
ENAL