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

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.


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 2 Evidence-Based Complementary and Alternative Medicine 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.

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.

Sample Analysis (Liquid Chromatography-Mass Spectrometry (LC-MS)).
Chromatography was performed in an ultra-performance liquid chromatography (UPLC6 Waters), using a reversed phase column HSS T3 C18 column, with 100 × 2.1 mm and 1.7 m 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-H 2 O (1 : 1, v/v), with injections of 5 L.
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.

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 22 ± 2 ∘ C under a 12/12 h light dark cycle, 55 ± 10% 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.

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.
Evidence-Based Complementary and Alternative Medicine 3 Only hypertensive rats (SBP above 140 mm Hg) were used in experiments.

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 (Roche5 Cobas Integra 400 plus). Excretion load (El) of Na + , K + , Cl − , and Ca ++ was obtained according to the equation El = × , where is the concentration of electrolytes (mEq/L) and is the urinary flow (mL/min). Results were expressed as Eq/min/100 g.

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 PowerLab5 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.

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; CaCl 2 2.4; MgSO 4 1.2; NaHCO 3 25.0; KH 2 PO 4 1.2; dextrose 11.1; and EDTA 0.03) gassed with 95% O 2 /5% CO 2 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 60minute equilibration period, MVBs were continuously perfused with PSS containing 3 M 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 l.

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]. Evidence-Based Complementary and Alternative Medicine cyclooxygenase inhibitor) and L-NAME (100 M; 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.

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% ( < 0.05), and results are expressed as mean ± standard error of the mean (SEM). Graphs were drawn and statistical analysis was carried out using GraphPad5 Prism software version 5.0 for Mac OS X (GraphPad Software, San Diego, CA, USA).

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].
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 As expected, animals that received HCTZ had a significant increase in urinary volume only on the 1st day of treatment.
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).

Prolonged Treatment with ESAH Restores Vascular Reactivity in MVBs from 2K1C OVT-Rats.
In MVBs from 2K1C plus OVT-rats, the administration of ACh or SNP was able to induce a vasodilatory response approximately 35% lower than that in Sham-operated rats (Figures 3(a) and 3(b)). Similarly, the vasoconstrictor response of PHE was significantly higher in the 2K1C plus OVT-group when compared to Shamoperated animals (Figure 3(c)). On the other hand, in animals that received ESAH (30 mg/kg) or ENAL (15 mg/kg), the effects of ACh, SNP, or PHE were not different from those observed in Sham-operated animals. Treatments with HCTZ (25 mg/kg) or ESAH at doses of 100 and 300 mg/kg were not able to reverse the changes in vascular reactivity observed in 2K1C plus OVT-animals (Figures 3(a)-3(c)). On the other hand, only ENAL was able to reduce the aldosterone levels and serum ACE activity (Figures  4(a)-4(c)), without affecting serum vasopressin. Treatment with ESAH (30, 100, and 300 mg/kg) or HCTZ did not significantly change the aldosterone and vasopressin concentrations or serum ACE activity (Figures 5(a)-5(c)).

Discussion
In the present study, it was demonstrated that 28-day ESAH treatment was effective in inducing an important saluretic and antihypertensive response in 2K1C plus OVT-rats. This favorable effect induced by ESAH is associated with a parallel     reduction of oxidative and nitrosative stress biomarkers, in addition to a possible increase in NO bioavailability. Additionally, an important NO and prostaglandin-dependent vasodilator effect was observed in mesenteric vascular bed, a finding that indicates a potential mechanism for the cardiovascular effects of ESAH.
In the first stage of our study, we decided to use ENAL and HCTZ as standard cardioprotective drugs. The choice of HCTZ was based on the need to compare the diuretic and natriuretic effects of ESAH with a first-line diuretic drug in the treatment of hypertension [17]. On the other hand, as the renovascular hypertension model used in our study (2K1C) induces a sustained activation of the renin-angiotensin system (RAS), we chose another classic drug (ENAL) capable of effectively blocking activation and preventing the installation of renovascular hypertension. In addition, data indicated that  ACE inhibitors, including ENAL, would exert cardioprotective effects regardless of the hypotensive action [18]. Current findings have indicated that ACE inhibitors may increase NO bioavailability and reduce oxidative and nitrosative stress parameters during hypertension. These effects provide an additional cardioprotective response to the sustained antihypertensive effect [19]. Therefore, considering our findings, ENAL was presented as a standard drug for comparison with the cardioprotective potential of ESAH.
Another point that deserves attention is the initiation of treatments with ESAH, ENAL, and HCTZ only 4 weeks after the surgical procedure. This option stems from the need to submit animals to a previous period of sustained activation of RAS and estrogen deprivation, a limiting step in oxidative/nitrosative imbalance and in the establishment of hypertension. In fact, all 2K1C plus OVT-rats had a high degree of oxidative/nitrosative stress, accompanied by increased vascular reactivity to PHE and a reduced vasodilatory    When we look closely at the renal effects of ESAH, we will see that while HCTZ showed an expected diuretic response, with an increase in water and electrolyte elimination after first administration, followed by a reduction of the diuretic effect by compensatory mechanisms, ESAH did not affect the urine volume eliminated. On the other hand, an important saluretic effect was evident, especially on the elimination of Na + and Cl − throughout the experimental period. In fact, at the lowest doses (30 and 100 mg/kg), the saluretic effect was evident only on the 1st and 7th days of treatment, while renal Na + and Cl − elimination continued significantly only at the highest dose tested (300 mg/kg). If we look at the antihypertensive effect of ESAH, we will see that the most significant reduction in blood pressure levels also occurred at the lowest dose used. This fact may explain in part the reduction of the saluretic response observed after the 7th day of treatment, since an eventual reduction of renal hydrostatic pressure, due to the sustained reduction of blood pressure, can reduce glomerular filtration rate and renal salt and water elimination. This effect was also evident with HCTZ, a classic antihypertensive drug that, despite inducing significant diuretic effect in the first dose, showed an important reduction in its efficacy after prolonged treatment.
It is known that cardiorenal regulation may involve several endogenous mediators, including prostaglandins, NO, and the endothelium-derived hyperpolarizing factor, synergistically acting in a complex hemodynamic and neurohumoral interaction [20]. In our study, it has been shown that the cardiorenal effects induced by ESAH appear to be influenced by an expressive antioxidant activity, which could influence the increase in the bioavailability of NO and consequently the vascular reactivity. Although suggestive, there is still some doubt whether the effect is due to direct release of NO by secondary metabolites present in ESAH or a consequence of the potent antioxidant activity. The fact is that substances that reduce reactive oxygen species may increase the bioavailability of NO and prostaglandins and consequently induce systemic vasodilator responses [21].
In order to prove this hypothesis, we evaluated the ability of ESAH to reduce perfusion pressure in the MVBs from normotensive and 2K1C plus OVT-rats in the absence and presence of L-NAME and indomethacin, two drugs capable of inhibiting NO and prostaglandin synthase, respectively. The putative blockade of NO and prostaglandin synthesis prevented the reduction of perfusion pressure induced by ESAH 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 that reinforces our findings was described in a recent study [5] where it shows that an extract with a similar phytochemical profile was able to induce a NO-dependent acute hypotensive response in normotensive rats, evidencing the importance of NO in the cardiovascular effects of ESAH. Although we believe that the cardiorenal effects presented in this study result from a complex interaction among different secondary metabolites present in the ESAH, the fact that there may be one or more agents involved cannot be ruled out. Biomonitoring studies can help answer these questions and, if possible, identify the agent that stands out in this process.

Conclusions
A 28-day ESAH treatment reduces the blood pressure levels in 2K1C-ovariectomized rats. These effects are associated with an important antioxidant and antinitrosant action, in addition to a significant saluretic effect. Probably, this response has a direct contribution of the reduction in vascular resistance, possibly mediated by the release of NO and prostaglandins.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.