Polyphenolic Composition and Hypotensive Effects of Parastrephia quadrangularis (Meyen) Cabrera in Rat

Parastrephia quadrangularis (Pq), commonly called “Tola”, is widely used in folk medicine in the Andes, including for altitude sickness. In this study, polyphenolic composition was determined, and hypotensive effects were measured; the ethnopharmacological use as hypotensive was related to the presence of phenolic compounds. For this purpose, male Sprague-Dawley rats (6 to 8 weeks of age, 160 to 190 g) were fed Pq extract (10 to 40 mg/kg) for 10 days through gavage. Blood pressures and heart rate were significantly (p < 0.01) reduced in normotensive rats receiving Pq extract (40 mg/kg body weight). Pq extract induced a negative inotropic effect, and endothelium-dependent vasodilation mediated by nitric oxide (NO). Furthermore, preincubation with Pq extract significantly decreased the cytosolic calcium on vascular smooth muscle cells A7r5 in response to L-phenylephrine (PE). Seven metabolites were isolated from the Pq extract, but three flavonoids (10−4 M) showed similar vasodilation to the extract in intact rat aorta as follows: 5,3′,4′-trihydroxy-7-methoxyflavanone (2); 3,5,4′-trihydroxy-7,8,3′-trimethoxyflavone (6); and 5,4′-dihydroxy-3,7,8,3′-tetramethoxyflavone (7). The Pq extract and compounds 2 and 7 significantly (p < 0.05) reduced the contraction to Bay K8644 (10 nM, an agonist of CaV1.2 channels). Administration of Pq decreased cardiac contractility and increased endothelium-dependent and -independent vasodilation.


Introduction
Parastrephia quadrangularis (Meyen) Cabrera, is a native shrub belonging to the Asteraceae family, found in the Northern Andes of Chile, Argentina, Southern Perú, and Bolivia. The plant heights are in the range of 0.3-1.5 m and several plants grow together forming a "piso puneño" or "tolar", a green

Plant Material
The plant material (branches, leaves, and inflorescences) from P. quadrangularis was collected from the Antofagasta region of Chile (22 • 19 31.80 S y 68 • 00 22.20 W, at 4000 m above the sea level, November 2015), and was subsequently identified and stored with a voucher number: PQ20151115.

Extract Preparation
The specimens were then dried and mechanically grounded to fine powder to exhaustively extract the principles to use in the pharmacological study (all procedures performed at room temperature 25 • C). A mass of 1.5 kg of the dry and powdered plant was deposited into a cotton bag with 3 L of a mixture EtOH:H 2 O (1:1) for 72 h inside a glass beaker at room temperature. Then, Whatman (filter paper) was used to filter the resulting solution; a rotary evaporator (50 • C) was subsequently used to evaporate the ethanol. The resulting aqueous extract was freeze-dried with a Labconco 4.5 FreeZone lyophilizer. The total extract yield was about 26%, which was then stored at 4 • C.

UHPLC-DAD-MS Instrument
A Thermo RS 3000 Q exactive focus was used as an System Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS; Thermo Fisher Scientific, Bremen, Germany), following methods as described by Simirgiotis [22]. Briefly, 10 µL of mixture (containing 2 mL methanol and 5 mg of our extract) was injected into the instrument following filtration using Polytetrafluoroethylene (PTFE) 200 µm filters.

LC and MS Parameters
An Acclaim UHPLC C18 column at 25 • C Thermo Scientific equipment manufactured in Bremen, Germany) (150 mm × 4.6 mm ID, 2.5 µm, was used for the analysis. The wavelengths were set at 330, 254, 354, and 280 nm, and the photodiode array detector was used from 800 to 200 nm. Aqueous and mobile phases were observed 1% formic acid solutions. The gradient employed was: 0.00 min, 5% B; (5.00 min, 5% B; 10.00 min, 30% B; 15.00 min, 30% B; 20.00 min, 70% B; 25.00 min, 70% B; 35.00 min, 5% B) and lastly, before each injection (at volume 10 µL), a waiting time of 12 min for equilibration, at 1.00 mL min −1 flow rate. The resin extract and standard compounds were incubated at 10 • C in the autosampler before the injections. Detection of all compounds was performed using a Q-Exactive Orbitrap mass spectrometer at 17,500 Full Width at Half Maximum (FWHM) (m/z 200), and the HESI II probe values were optimized as previously described [22,23].

Animals
To evaluate the traditional use of the plant, 37 male Sprague-Dawley rats (6 to 8 weeks of age, 160 to 190 g) from the Antofagasta University breeding colony were randomly assigned into the following groups: Group 1 (n = 5) was used for measurement of blood pressure after intravenous bolus of Pq. In this group, Pq extract was acutely injected (intravenously) in cumulative doses (10 to 80 mg/kg) in the same rats after recovery of basal pressure. Groups 2 to 5 received gavage, vehicle or Pq extract. Group 2 was used as control, and only received vehicle (saline solution). Groups 3 to 5 (n = 5) were used for measurement of blood pressure after oral treatment with Pq for 10 days (10, 20, and 40 mg/kg body weight Pq). Group 6 (n = 7) for Langendorff was used for preparation and group 7 (n = 5) was used for vascular reactivity experiments. Animals had access to food (standard rat chow from Champion, Santiago, Chile) and water ad libitum, in a temperature and light controlled room. Experiments were in accordance to institutional (Universidad Antofagasta Ethics Committee, CEIC 135/2018) and use of laboratory animal care (National Institutes of Health, revised 2013).2.8. Measurement of Blood Pressure.
Blood pressure was measured on the rats with procedures previously described [24]. The rats were anesthetized with xylazine (5 mg/kg, i.p.) and ketamine (42 mg/kg, i.p.). Following the method as described above, we measured blood pressure in vivo using a pressure transducer TSD 120 connected to a DA100B amplifier (Biopac Systems Inc, Santa Barbara, CA USA). AcqKnowledge III systems software v3.9.1.6 (Santa Barbara, CA, USA) was used for blood pressure recording and data analysis.

Vascular Reactivity Experiments
Aortic rings from the same animal were concurrently studied in different organ baths [25] for comparable abilities and reactivity function. After a 30 min period of equilibration, the aortic rings were stabilized with KCl (mM) near-maximum contractions for 10 min. We maintained a passive tension of 1.0 g on the aorta, which was determined to be the optimal resting tension for obtaining maximum active tension [26] in our laboratory.
Vasodilation to 10 −5 M ACh (muscarinic agonist) in aortic rings pre-contracted with 10 −6 M PE was used as a method to assess endothelial function. Functionality was confirmed with a vasodilation of 70% to 80% [27]. Vascular reactivity of the extracts was observed with the addition of pure and crude compound dilutions into the organ bath following rings pre-contracted with 10 −6 M PE, or preincubation with Pq for 20 min, then contraction with 10 −6 M PE.
In some experiments, a similar protocol was repeated in the absence of endothelium (endothelial removal was performed by gently rubbing the inner lining using a small piece of cotton), or in presence of N w -nitro-L-arginine methyl ester (L-NAME, 10 −4 M).

Cytosolic Calcium Signal on Vascular Smooth Muscle Cells
Vascular smooth muscle cell line A7r5 (ATCC CRL-1444) were cultured in cover slips and treated with 10 µM Fluo-3 AM (Thermo Fisher Scientific Waltham, MA, USA) in KRB for 30 min at 37 • C. A Carl Zeiss LSM-5 Pascal 5 fluorescence Axiovert 200 microscope was used to study the cells at 527 nm. Cells were pretreated for 20 min with Pq (100 µg/mL) or vehicle, and then stimulated with PE 10 −6 M. Images were collected every 1 s and analyzed with ImageJ software (v1.8.0_112, Bethesda, MD, USA, NIH). Cytosolic Ca 2+ is expressed as ∆F/F 0 (relative fluorescence).

Determination of Antioxidant Activity
In vitro, antioxidant activity was determined using the methods described by Larrazabal-Fuentes et al. 2019 [28] in Supplementary File 1 (Supplementary Materials). The absorbance of each assay was determined in a microplate reader (BioTek Synergy HTX Multimodal equipment; Winooski, VT, USA).

Statistical Analysis
Data were expressed as average ± standard error (SEM). A Bonferroni post hoc test was performed following a two-way analysis of variance (ANOVA) between dose-response curves. IC 50 was calculated by nonlinear regression (sigmoidal) and p < 0.05 was considered statistically significant. Graph Pad PrismTM software, version 5.0. (GraphPad Software, Inc., La Jolla, CA, USA) was used.

The Hydroalcoholic Extract from P. quadrangularis Causes a Hypotensive Effect in Rats
To evaluate the hypotensive effect of extract on blood pressure, we intravenously injected a bolus of different doses of Pq (10 to 80 mg/kg bw).
It is common for hypotensive substances to cause a rapid vasodilator effect, decrease total peripheral resistance, and a compensatory effect of the cardiovascular system recovers normal blood pressure through increased cardiac output and heart rate, as a reflex effect ( Figure 1). Vascular smooth muscle cell line A7r5 (ATCC CRL-1444) were cultured in cover slips and treated with 10 μM Fluo-3 AM (Thermo Fisher Scientific Waltham, MA, USA) in KRB for 30 min at 37 °C. A Carl Zeiss LSM-5 Pascal 5 fluorescence Axiovert 200 microscope was used to study the cells at 527 nm. Cells were pretreated for 20 min with Pq (100 μg/mL) or vehicle, and then stimulated with PE 10 −6 M. Images were collected every 1 s and analyzed with ImageJ software (v1.8.0_112, Bethesda, MD, USA, NIH). Cytosolic Ca 2+ is expressed as ΔF/F0 (relative fluorescence).

Determination of Antioxidant Activity
In vitro, antioxidant activity was determined using the methods described by Larrazabal-Fuentes et al. 2019 [28] in Supplementary File 1 (Supplementary Materials). The absorbance of each assay was determined in a microplate reader (BioTek Synergy HTX Multimodal equipment; Winooski, VT, USA).

Statistical Analysis
Data were expressed as average ± standard error (SEM). A Bonferroni post hoc test was performed following a two-way analysis of variance (ANOVA) between dose-response curves. IC50 was calculated by nonlinear regression (sigmoidal) and p < 0.05 was considered statistically significant. Graph Pad PrismTM software, version 5.0. (GraphPad Software, Inc., La Jolla, CA, USA) was used.

The Hydroalcoholic Extract from P. quadrangularis Causes a Hypotensive Effect in Rats
To evaluate the hypotensive effect of extract on blood pressure, we intravenously injected a bolus of different doses of Pq (10 to 80 mg/kg bw).
It is common for hypotensive substances to cause a rapid vasodilator effect, decrease total peripheral resistance, and a compensatory effect of the cardiovascular system recovers normal blood pressure through increased cardiac output and heart rate, as a reflex effect ( Figure 1). Thus, we found that Pq extract caused a dose-dependent reduction in the mean arterial pressure (MAP) of normotensive rats (Table 1).  Thus, we found that Pq extract caused a dose-dependent reduction in the mean arterial pressure (MAP) of normotensive rats (Table 1). In the following experiments, doses between 10 and 40 mg/kg of the extract were administered through a gastric gavage for 10 days. Pq extract significantly reduced the mean arterial pressure (87 ± 5 mmHg control vs. 66 ± 1 mmHg with 40 mg/kg Pq, p < 0.01, n = 5, Figure 2A), the heart rate (347 ± 9 bpm control vs. 297 ± 6 bpm with 40 mg/kg Pq, p < 0.01, Figure 2B), and the diastolic blood pressure (DBP) in normotensive rats (75 ± 6 mmHg control vs. 49 ± 1 mmHg with 40 mg/kg Pq, p < 0.01, Table 2).
Values are mean ± standard error of the mean of 5 experiments in mmHg. ** p < 0.01 and *** p < 0.001 vs. control.
In the following experiments, doses between 10 and 40 mg/kg of the extract were administered through a gastric gavage for 10 days. Pq extract significantly reduced the mean arterial pressure (87 ± 5 mmHg control vs. 66 ± 1 mmHg with 40 mg/kg Pq, p < 0.01, n = 5, Figure 2A), the heart rate (347 ± 9 bpm control vs. 297 ± 6 bpm with 40 mg/kg Pq, p < 0.01, Figure 2B), and the diastolic blood pressure (DBP) in normotensive rats (75 ± 6 mmHg control vs. 49 ± 1 mmHg with 40 mg/kg Pq, p < 0.01, Table 2).  To study the effect of Pq extract on cardiac contractility, Langendorff was used. We confirmed that perfusion of the heart with the extract caused a dose-dependent negative inotropic effect. Although the maximal rate of increase (dP/dtmax) of left ventricular pressure did not decrease significantly with 100 μg/mL of the extract ( Figure 3B), the left ventricular pressure (LV pressure) was drastically reduced with 100 μg/mL of the extract (75 ± 4 mmHg basal vs. 51 ± 1 mmHg with 100 μg/mL Pq, p < 0.05, Figure 3A). The dose of 1000 μg/mL also reduced (p < 0.05) the contractility (dP/dtmax). The coronary perfusion pressure was stable in the presence of increasing doses of the extract ( Figure 3C). Perfusion of the isolated heart with KHB buffer did not recover the baseline contractility value (dP/dtmax), nor the left ventricular pressure, suggesting that effect of Pq remains. This result of washing with buffer was similar to that published with Senesio nutans or Xenophylum popusum [24,29].  To study the effect of Pq extract on cardiac contractility, Langendorff was used. We confirmed that perfusion of the heart with the extract caused a dose-dependent negative inotropic effect. Although the maximal rate of increase (dP/dt max ) of left ventricular pressure did not decrease significantly with 100 µg/mL of the extract ( Figure 3B), the left ventricular pressure (LV pressure) was drastically reduced with 100 µg/mL of the extract (75 ± 4 mmHg basal vs. 51 ± 1 mmHg with 100 µg/mL Pq, p < 0.05, Figure 3A). The dose of 1000 µg/mL also reduced (p < 0.05) the contractility (dP/dt max ). The coronary perfusion pressure was stable in the presence of increasing doses of the extract ( Figure 3C). Perfusion of the isolated heart with KHB buffer did not recover the baseline contractility value (dP/dt max ), nor the left ventricular pressure, suggesting that effect of Pq remains. This result of washing with buffer was similar to that published with Senesio nutans or Xenophylum popusum [24,29].   . shows the vasodilation dose-response curves in endothelium denuded and intact rings, or rings preincubated with L-NAME for the Pq-induced relaxation (−3 to 3 [log μg/mL], which is equivalent to 0.001 to 1000 μg/mL) As shown in Figure 4A, relaxation was lower in endotheliumdenuded aortic rings in the presence of Pq extract (58 ± 7% in control vs. 29 ± 9% in endotheliumdenuded aorta, 2 [log μg/mL] or 100 μg/mL, p < 0.001, n = 5). To understand the role of NO in mechanisms associated with Pq activity, we employed the use of endothelial pharmacological modulators.  Figure 4 shows the vasodilation dose-response curves in endothelium denuded and intact rings, or rings preincubated with L-NAME for the Pq-induced relaxation (−3 to 3 [log µg/mL], which is equivalent to 0.001 to 1000 µg/mL) As shown in Figure 4A, relaxation was lower in endothelium-denuded aortic rings in the presence of Pq extract (58 ± 7% in control vs. 29 ± 9% in endothelium-denuded aorta, 2 [log µg/mL] or 100 µg/mL, p < 0.001, n = 5). To understand the role of NO in mechanisms associated with Pq activity, we employed the use of endothelial pharmacological modulators.   Figure 4. shows the vasodilation dose-response curves in endothelium denuded and intact rings, or rings preincubated with L-NAME for the Pq-induced relaxation (−3 to 3 [log μg/mL], which is equivalent to 0.001 to 1000 μg/mL) As shown in Figure 4A, relaxation was lower in endotheliumdenuded aortic rings in the presence of Pq extract (58 ± 7% in control vs. 29 ± 9% in endotheliumdenuded aorta, 2 [log μg/mL] or 100 μg/mL, p < 0.001, n = 5). To understand the role of NO in mechanisms associated with Pq activity, we employed the use of endothelial pharmacological modulators.  The inhibition of nitric oxide synthase (NOS) with 10 −4 M L-NAME significantly (p < 0.001) blunted the relaxation of Pq. However, 1H-(1,2,4) oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of soluble guanylyl cyclase) significantly increased (p < 0.001) the Pq-induced vasodilation in aortic rings versus control ( Figure 4C). The IC 50 to Pq was significantly raised (p < 0.001) in the absence of endothelium and in the presence of L-NAME, while the preincubation with ODQ significantly (p < 0.001) decreased the IC 50 to Pq versus control (Table 3).

Role of Potassium Channels on Vasodilation of P. quadrangularis (Pq)
At Pq extract concentrations of 2 [log µg/mL] or 100 µg/mL and 3 [log µg/mL] or 1000 µg/mL, there were significant differences with respect to the control, where relaxation was lower in the presence of the 10 −5 M BaCl 2 , 10 −5 M glibenclamide, and 1 mM tetraethylammonium (TEA, p < 0.001, Figure 5). The IC 50 to Pq was significantly (p < 0.001) raised in the presence of BaCl 2 , and TEA versus control (Table 3 and Figure 5).

P. quadrangularis (Pq) Reduced the Contractile Response to KCl and PE
The effect of Pq extract on contractile response to KCl and PE in aortic rings of the rat was studied. The preincubation with Pq extract (100 µg/mL) significantly (p < 0.001) reduced the maximal contraction to 60 mM KCl (125 ± 3% control vs. 52 ± 11%, Figure 6A), and the maximal contraction in response to 10 −6 M PE (145 ± 4% for control vs. 85 ± 8%, with 100 µg/mL of Pq, p < 0.001). The EC 50 to PE was significantly (p < 0.01) increased in the presence of Pq extract versus control (Table 4). Nimodipine was used to compare if Pq extract is a blocker of L-type voltage-gated Ca 2+ channels. In this sense, 10 −4 M nimodipine significantly (p < 0.001) reduced the maximal contraction to 60 mM KCl (125 ± 3% control vs. 5 ± 2% with nimodipine, Figure 6A) and 10 −6 M PE (156 ± 5% control vs. 57 ± 7% with nimodipine, Figure 6B). Interestingly, with 100 µg/mL Pq preincubation, there was a decrease in the cytosolic calcium signal on vascular smooth muscle cell line A7r5 response to 10 −6 M PE stimulation ( Figures 6C and 7).
In the following experiments, we compared the relaxation and blocking of Cav1.2 channels by Pq, flavonoids 2 and 7, and quercetin. On the one hand, Pq extract, the flavonoids 2 and 7 (p < 0.01), and quercetin (p < 0.05) reduced the contractile response to 15 mM KCl ( Figure 9B). On the other hand, quercetin did not decrease the contractile response to 10 −8 M Bay K8644, an agonist of CaV1.2 channels, versus control ( Figure 9C).
In the following experiments, we compared the relaxation and blocking of Cav1.2 channels by Pq, flavonoids 2 and 7, and quercetin. On the one hand, Pq extract, the flavonoids 2 and 7 (p < 0.01), and quercetin (p < 0.05) reduced the contractile response to 15 mM KCl ( Figure 9B). On the other hand, quercetin did not decrease the contractile response to 10 −8 M Bay K8644, an agonist of Ca V 1.2 channels, versus control ( Figure 9C).

Determination of the Antioxidant Content of P. quadrangularis (Pq)
Results from the quantitative determination of in vitro antioxidant activity for Pq are summarized in Table 5. The quantification of the content of phenolic compounds and flavonoid in Pq demonstrated the extract that contained the highest amount of polyphenols, i.e., 482 ± 19 mg gallic acid equivalent/g extract and a moderated value of flavonoids with 140 ± 4 mg quercetin equivalent/g extract, respectively. The nitric oxide radical quenching activity of the Pq was detected and compared with the standard ascorbic acid. The extract exhibited a low capacity to inhibit the nitric oxide radical, with an IC50 value of 498 ± 5 μg/mL in a concentration-dependent manner. Ascorbic acid inhibited the nitric oxide radical, with an IC50 value of 48 ± 1 μg/mL, the latter being 10 times more effective.

Determination of the Antioxidant Content of P. quadrangularis (Pq)
Results from the quantitative determination of in vitro antioxidant activity for Pq are summarized in Table 5. The quantification of the content of phenolic compounds and flavonoid in Pq demonstrated the extract that contained the highest amount of polyphenols, i.e., 482 ± 19 mg gallic acid equivalent/g extract and a moderated value of flavonoids with 140 ± 4 mg quercetin equivalent/g extract, respectively. The nitric oxide radical quenching activity of the Pq was detected and compared with the standard ascorbic acid. The extract exhibited a low capacity to inhibit the nitric oxide radical, with an IC 50 value of 498 ± 5 µg/mL in a concentration-dependent manner. Ascorbic acid inhibited the nitric oxide radical, with an IC 50 value of 48 ± 1 µg/mL, the latter being 10 times more effective.
The ferric reducing/antioxidant power (FRAP) assay is based in the reduction of Fe +3 to Fe +2 in the presence of TPTZ (2,4,6-tris-(2-pyridyl)-s-triazine) and an antioxidant agent, thus, forming an intense complex of blue Fe-TPTZ. The FRAP assay result of the Pq shows that the extract possessed a high reducing power with 760 ± 12 mg trolox equivalent/g extract. The extract provided an antiradical activity dose-dependently inhibiting the radical 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) with an IC 50 value of 201 ± 4 and 127 ± 5 µg/mL, respectively. The inhibitory activity was approximately three times lower than the trolox standard for the DPPH radical with an IC 50 value of 61 ± 3 µg/mL and two times lower for the radical ABTS, which showed IC 50 values of 77 ± 2 µg/mL, respectively. The total antioxidant potential of the extract was estimated in phosphomolybdate and hexacyanoferrate assay, the extract exhibits a high reduction capacity with values of IC 50 of 115 ± 4 µg/mL and 73 ± 2 µg/mL, respectively. The standard ascorbic acid shows values of 23 ± 1 µg/mL and 19 ± 3 µg/mL, in both methods studied.

Coumarins
Using co-elution procedures with already identified samples, we were able to identify peaks 10 and 13 as umbelliferone and scopoletin (3). Subject to further confirmation, peak 12 was identified as the dicoumarin euphorbetin because of its parent deprotonated ion at m/z 353.02919 [35,36] and peak 8 with a parent deprotonated molecule at m/z 515.08313 as its derivative euphorbetin glucoside (C 24 Figure S1 (Supplementary Materials).

Flavonoids
Peak 20 was identified as isorhamnetin (C 16 H 11 O 7 − ) because of the pseudomolecular ion at m/z 315.05090 [34]. Its identity was confirmed using co-injection with an authentic standard, and peak 18 and 19 as the flavonols kaempferol and quercetin, while peaks 23 and 24 as their derivatives 7-methoxykaempferol and (C 16

Discussion
The hypotensive effects, and possible mechanisms, are involved in the ethnopharmacological uses of P. quadrangularis (crude and purified compounds) for treatment of cardiovascular complications. In folk medicine in northern Chile, Parastrephia quadrangularis concotions are mainly drunk as herbal teas, water infusions, or decoctions [3]. Therefore, scientific investigations and validation are useful for the preparation of nutritional supplements [41] or pharmacological preparations.
This study suggests that the hydroalcoholic extracts from Pq possess a negative inotropic effect, especially on its effects on calcium availability, and the possibility of reductions in peripheral resistance and blood pressure. The decrease of diastolic pressure could be through an alpha-adrenergic receptor blocker [42] and vasodilator effects, leading to a reduction in systemic vascular resistance.
Forty-three compounds including several tremetones, coumarins, and flavonoids were identified in the hydroalcoholic extract, and several of those compounds were isolated and tested regarding their hypotensive effects.
Results show that the negative inotropic effect and the reduction of heart rate observed could cause the drop of blood pressure and suggest a direct cardiac modulation by Pq extract [29], and that the effects of Pq in reducing vascular contractile response on endothelium-intact rings occurs in a dose-dependent manner. In fact, the absence of the endothelium or the inhibition of the NOS with L-NAME significantly reduced the Pq-induced vasodilation in aorta. Traditionally, vasoactive substances cause vasodilation either by stimulating the NO/sGC pathway, activating the potassium channels, or blocking the Cav1.2 channels [43]. However, the inhibition of guanylate cyclase soluble (sGC) with ODQ did not blunt the Pq-induced vasodilation, suggesting another mechanism involved, different to the NO/sGC pathway.
Schinzari et al. [44] reported that potassium channel activation in vascular smooth muscles leads to the hypolarization of the vascular membrane, and an increase in vasodilation. The preincubation with BaCl 2 , glibenclamide, and TEA significantly diminished the Pq-induced relaxation.
Another possibility may be that Pq extract produced vasodilation through a modulation of influx of calcium from extracellular sources through Ca V 1.2 [45]. This observation was confirmed by subsequent experiments. First, the preincubation with Pq reduced the contractile response mediated by the membrane depolarization with KCl and pharmacological stimulation with PE. Secondly, the effects of preincubation with Pq and nimodipine (nonselective blocker of L-type voltage-gated Ca 2+ channels) on PE induced contractile responses to the aortic rings were alike. Thirdly, there was a decrease in the A7r5 vascular smooth muscle cell line cytosolic calcium with PE following incubation with Pq, which was also observed with Bay K8644 that acts through Cav1.2 channels on intact aortic rings.
To evaluate the vasodilation effect of the seven pure isolated compounds, a screening test was conducted in intact aortic rings of rats.
These findings strongly suggest that the hypotensive activity described above could be explained by a decrease in peripheral resistance. Notwithstanding the fact that vessels like the aortas are conductance vessels, and play little roles in peripheral resistance, the vascular reactivity properties of the extract shows that it could reduce peripheral resistance through a reduction in vascular myogenic tone. This is in agreement with our results and is complementary to the negative inotropic effects seen in the cardiac musculature of the heart.
The extract Pq exhibited moderate-high antioxidant properties, with phenolic and flavonoid constituents, and similar vascular abilities following pre-contraction with agonists like Bay K8644 (an agonist of Ca V 1.2 channels) and PE. These data agree with previous studies of different species of Parastrephia (P. lepidophylla, P. lucida, and P. phyliciformis (Meyen) Cabrera) [13].
Three isolated flavonoids caused a reduction of the contractile vascular response to influx of extracellular Ca 2+ and was likely mediated by blockage of Cav1.2 channels. In addition, Pq extract reduced the contractile response to PE in a similar way at nimodipine (a nonselective blocker of L-type voltage-gated Ca 2+ channels).
Vascular relaxation observed by the extract and three flavonoids isolated from Pq, which was similar to ACh-induced endothelial vasodilation via endothelial NO, strongly suggest that antioxidant activity of Pq and its metabolites may be involved. We hypothesize that three isolated flavonoids would increase the bioavailability of endothelial NO, leading to endothelial vasodilation. In contrast to flavonoids 2, 6, and 7 isolated from Pq, quercetin, a standard flavonoid with a high antioxidant activity, did not cause a significant vasodilation. This result would indicate that quercetin exerts a cardio-protector effect by other pathways, such as preventing the lipopolysaccharide-induced oxidative stress, or reducing lipid peroxidation and protein oxidation [46].

Conclusions
In conclusion, P. quadrangularis demonstrated hypotensive ability in normotensive animals; the mechanisms involved include an increase in vasodilation response, a decrease in heart rate, and cardiac contractility via inotropic effects. The mechanisms associated with these effects include endothelium-dependent vasodilation by NO and independent mechanisms, an activation of the potassium channels, and a decrease in cytosolic calcium.