The Hydroalcoholic Extract of Leaves of Mandevilla moricandiana Induces NO-Mediated Vascular Relaxation

• Abstract
• Introduction
• Results
• Discussion
• Materials and Methods
• Plant material and extraction
• HPLC-DAD analysis for flavonoids quantification
• LC-ESI-MS and ion trap LC-MS/MS analysis
• Total flavonoid content
• Preparation of aortic rings
• Chemicals and drugs
• Statistical analysis
• Supporting information
• Acknowledgements
• References

Abstract

Natural products extracted from plants represent a valuable source of new bioactive substances. Many studies describe the potential of plant products for the treatment of cardiovascular diseases. Species of the Mandevilla genus have been studied for their biological activities, mainly as antioxidant, anti-inflammatory, and vasorelaxant. However, the phytochemical and pharmacological profiles of Mandevilla moricandiana have not been investigated yet. The aim of this study was to evaluate the vasodilator effect of the hydroalcoholic extract of the leaves of M. moricandiana, as well as its chemical profile. Chemical analysis and quantification of major compounds were performed by HPLC analysis. Total flavonoid content was quantified based on rutin equivalents, and major compounds were identified based on HPLC-DAD-MS analysis. M. moricandiana leaf extract-induced vasodilation was investigated in rat aortic rings precontracted with phenylephrine. The total flavonoids were quantified as 3.25 ± 0.11 % w/w of the hydroalcoholic leaf extract, and HPLC-DAD-MS allowed for the identification of luteolin and quercetin glycosides. The maximal relaxant effect of the hydroalcoholic leaf extract was 86.07 ± 1.68 % at a concentration of 30 µg/mL (p < 0.05; n = 6). The concentration of hydroalcoholic extract of the leaves of M. moricandiana necessary to reduce phenylephrine-induced contractions of the endothelium-intact aorta by 50 % was 0.82 ± 0.10 µg/mL. M. moricandiana leaf extract-induced vasodilation was abolished in aortas pretreated with NG-nitro-L-arginine methyl ester and 1H-[1,2,4]oxadiazolo-[4,3-α]quinoxalin-1-one. In addition, diphenhydramine partially inhibited the effect of the hydroalcoholic extract of the leaves of M. moricandiana. Thus, M. moricandiana-induced relaxation depends on the endothelium and on the activation of the nitric oxide/cyclic GMP pathway, with the involvement of endothelial histamine H₁ receptors. Luteolin and quercetin glycosides seem to contribute to the extract activity.

Introduction

Several species of the genus Mandevilla, the largest genus in the Apocynoideae, a subfamily of the Apocynaceae family, have pharmacological activities described and the most studied are Mandevilla velutina K.Schum. and Mandevilla illustris (Vell.) Woodson. The hydroalcoholic extract of M. velutina rhizomes inhibits bradykinin-induced contractions of arterial and venous rabbit vessels [1], as well as of isolated guinea pig trachea [2]. Also, anti-edematogenic and antinociceptive [3] activities were described. Biondo et al. [4] have shown that the aqueous extract of M. illustris can fully inhibit the phospholipase activity of the crude venom of Crotalus durissus terrificus and partially inhibit the action of crotoxin of this snake. Others activities described for M. illustris were uterine relaxant [5] and anti-edematogenic [6].

Recently, Cordeiro et al. [7] characterized the chemical composition of the chloroform and hexane extracts in epicuticular wax from the leaves of Mandevilla moricandiana (A. DC.) Woodson, which have n-alkanes and the triterpene lupeol. However, no information is available regarding the pharmacological properties of this species. Therefore, the purpose of the present study was to investigate the effects and the mechanisms of action of ELM on rat vascular smooth muscle, as well as to evaluate its chemical profile.

Results

HPLC-DAD-MS analyses of ELM led to the identification of four major flavonoids (peaks with t_R at 18.3, 20.7, 22.6, and 23.3 min) at 265 nm ([Fig. 1]). Peaks with t_R at 18.3 and 22.6 min displayed a typical UV absorption of flavonol (typical λ _max 250–280 and 350–380 nm) ([Table 1]) [8]. The MS spectrum showed a pseudo-molecular ion at m/z 597 [M + H]⁺ in the positive ionization mode and fragmentation ions at m/z 465 and m/z 303, produced after elimination of 132u followed by the loss of 162u, indicating a disaccharide composed of pentose and glucose. The nature of the (1 → 2) interglycosidic linkage can be suggested by the evidence that ion at m/z 303 [(M + H)-294]⁺ is much more abundant than the ion at m/z 465 [(M + H)-132]⁺. This flavonoid was suggested as quercetin 3-O-pentosyl-glucoside. The flavonol at 22.6 min was identified based on UV and MS spectra as quercetin-3-glucuronyl-pentoside with a pseudo-molecular ion at m/z 611 [M + H]⁺ in the ESI+ mode and fragmentation ions at m/z 435 and 303 that correspond to the sequential loss of a glucuronyl and a pentose unit, respectively.

Peaks with t_R at 20.7 and 23.3 min correspond to flavones with maxima at 265 and 348 nm (typical λ _max 251–271 and 335–350 nm) [8], the first one being the major compound of ELM. The MS spectrum of the peak at 20.7 min showed a pseudo-molecular ion at m/z 581 [M + H]⁺ in the positive ionization mode and characteristic fragmentation ions at m/z 449 and m/z 287 due to the sequential loss of a pentose and glucose. Its structure was suggested as luteolin-7-O-pentosyl-glucoside. The second proposed flavone (t_R 23.3 min) was suggested as luteolin-7-O-glucoside, known as cynaroside, with a pseudo-molecular ion at m/z 449 [M + H]⁺ and fragmentation ion at m/z 287 due to the loss of a glucosyl unit. The presence of the aglycone luteolin for these flavones was proposed based on the MS-MS of the ion at m/z 287. There were observed at ions m/z 269 [M + H-H₂O]⁺, 241 [M + H-H₂O-CO]⁺, 231 [M + H-2CO]⁺, and 213 [M + H-2CO-H₂O]⁺ [9].

Rutin was also found as a constituent of the extract and corresponded to the peak observed at 19.7 min. The MS for this peak showed a compatible profile for rutin with a pseudo-molecular ion at m/z 611 [M + H]⁺ and fragmentation ion at m/z 465, corresponding to a loss of deoxyhexose (146u) and m/z 303 related to the aglycone quercetin. The sum of all flavonoids peaks, based on their UV spectrum, in the chromatogram was assumed to represent the total flavonoid content of the extract, expressed as rutin equivalents, the percentage (w/w) g/100 g of ELM. For this purpose, ELM was analyzed in triplicate, resulting in a flavonoid content of 3.25 ± 0.11 % w/w.

[Fig. 2] shows a typical recording of the maximal contractile response induced by 10 µM phenylephrine in the aorta with ([Fig. 2 A]) and without ([Fig. 2 B]) endothelium followed by exposure to cumulative concentrations of ELM. It elicited a concentration-dependent relaxation in the precontracted aorta with a functional endothelium. At 30 µg/mL, ELM produced a relaxation of 86.07 ± 1.68 % (p < 0.05; n = 6) ([Fig. 2 C]). The concentration necessary to reduce 50 % of the maximal contraction induced by phenylephrine (IC₅₀) was 0.82 ± 0.10 µg/mL. Removal of the endothelium completely inhibited ELM-induced vasorelaxation.

Pretreatment of the aortic rings with endothelium using L-NAME and ODQ produced a complete inhibition of the vascular relaxation induced by ELM ([Fig. 3 A]), indicating the involvement of the NO/cGMP pathway to the effect. In addition, pretreatment with TEA significantly inhibited the vasorelaxant response to ELM ([Fig. 3 A]). On the other hand, indomethacin did not significantly alter the vasodilation produced by ELM ([Fig. 3 B]).

In an attempt to determine the receptor involved in the activation of the NO-cGMP system, aortic rings were incubated with atropine, HOE-140, fulvestrant, and diphenhydramine. Activation of muscarinic and bradykinin receptors is not involved since atropine and HOE-140 did not modify the vasorelaxation produced by ELM ([Fig. 3 C]). Diphenhydramine induced a rightward displacement of the concentration-response curve of ELM at all concentrations tested ([Fig. 3 D]). In the presence of 10 µM diphenhydramine, ELM-induced relaxation at 30 µg/mL was reduced from 86.07 ± 1.68 % to 59.16 ± 5.21 % (p < 0.01; n = 6) and the IC₅₀ increased to 2.84 ± 0.32 µg/mL (p < 0.05). In addition, in the presence of fulvestrant, the IC₅₀ increased to 1.86 ± 0.32 µg/mL (p < 0.05), but without a reduction of the maximal relaxation ([Fig. 3 D]).

Discussion

The present study shows that ELM relaxes vascular smooth muscle via the endothelium-dependent eNOS-sGC pathway, which may be related, at least in part, to the activation of histamine receptors. This is the first report of the effects of M. moricandiana on vascular reactivity, which has not been previously investigated.

ELM-induced vasodilation was dependent on the main endothelium-derived relaxant factor NO, which produces vasodilation through the activation of sGC and the consequent formation of cGMP. This cyclic nucleotide activates protein kinase G, which promotes vascular smooth muscle relaxation by mechanisms such as the activation of intracellular Ca²⁺ uptake by the sarcoplasmatic reticulum, increased intracellular Ca²⁺ efflux, the membrane hyperpolarization through the opening of Ca²⁺-activated K⁺ channels, and the reduction in the sensitivity of the contractile machinery [10], [11]. Thus, the rightward shift of the concentration-response curve to ELM observed in the presence of TEA may indicate NO/cGMP-dependent activation of K⁺ channels.

Besides NO, prostacyclin can also be released from the endothelium after being synthesized through cyclooxygenase activity to produce smooth muscle relaxation via the adenylate cyclase pathway [12]. Pretreatment with indomethacin did not alter ELM-induced relaxation, suggesting that prostanoids may not contribute to the vascular effect.

A number of substances isolated from plants have already been shown to produce endothelium-dependent, NO-mediated vasorelaxation. These include polyphenolic compounds such as flavonoids and tannins, which may activate the formation of NO from eNOS or may enhance NO-mediated relaxation by scavenging the superoxide anions [13].

The synthesis of NO may be activated through the activation of endothelial receptors such as muscarinic (M₃) and histamine (H₁) receptors, which leads to eNOS activation by elevating the intracellular Ca²⁺ concentration and the binding of the Ca²⁺-calmodulin complex to eNOS [11], [14]. On the other hand, stimuli such as shear stress, insulin, and 17β-estradiol activate NO production in a Ca²⁺-independent manner through the phosphorylation of eNOS. PI3K plays a major role in NO production and mediates eNOS activation through activation of protein kinase B (Akt), which in turn phosphorylates eNOS on Ser1177 [14].

Preincubation of the aortic rings with atropine and HOE-140 did not modify the vasodilation induced by ELM, indicating that muscarinic and bradykinin receptors are not involved in the ELM effect. Different from M. moricandiana, M. velutina extract showed a selective bradykinin-antagonism action in rabbit vascular smooth muscle [1].

Diphenhydramine produced a rightward shift of the concentration-response curve for ELM, indicating that histamine receptors are involved in the ELM effect. Endothelial histamine H₁-receptor stimulation leads to NO and prostacyclin production, to changes in vascular permeability, and synthesis of platelet-activating factor [15]. However, prostacyclin seem not to be involved in the vasodilation induced by ELM, as described above. Similar histamine H₁-receptor-mediated endothelium-dependent relaxation has also been observed with some plant extracts and phenolic compounds, as with the aqueous extract of Cirsium japonicum (Thunb.) Fisch. ex DC. (Asteraceae) [16], black currant [17], and the lignan eudesmin [18] in the rat aorta.

Some polyphenols, such as resveratrol [19], and polyphenols present in red wine [20] and black tea [21] have phytoestrogenic activity and can activate ER on the vascular endothelium. Both ERα and ERβ are expressed in the endothelium, although ERα is the predominant form localized to the plasma membrane and involved in non-nuclear signaling [22], [23]. ERα activation triggers NO production through PI3K/Akt pathway activation followed by eNOS phosphorylation [23], [24]. However, estrogen receptors seem to play a minor role in the ELM effect.

Taking into account that flavonoids were detected in ELM, they could contribute to the vasodilatory activity found. Quercetin and luteolin glycosides (quercetin-3-O-pentosyl-glucoside, quercetin-3-glucuronyl-pentoside, luteolin-7-O-pentosyl-glucoside, and luteolin-7-O-glucoside) seem to be the main active substances responsible for the ELM vasodilator effect.

The vasorelaxation effect of quercetin has been demonstrated in several studies. Quercetin induces vasodilation through both endothelium-dependent and endothelium-independent ways and it can increase eNOS activity and NO production by elevating the endothelial intracellular Ca²⁺ concentration [25] and eNOS phosphorylation [26]. Mechanisms of quercetin induced-vasodilation can be different in different vascular fields [27], [28]. There are not pharmacological activities described for quercetin-3-O-pentosyl-glucoside and quercetin-3-glucuronyl-pentoside, but quercetin-3-O-glucoside was shown to induce relaxation of the rat aorta in an endothelium-independent manner [29].

Studies demonstrated that luteolin causes vasodilation of the rat aorta through the activation of eNOS by increasing its phosphorylation at Ser177 [30] and protects arteries from injury by oxidative stress [31]. On the other hand, in porcine coronary and splenic arteries, the relaxation response to luteolin is mediated through the inhibition of Ca²⁺ influx [32]. Different activities have already been shown for luteolin-7-glucoside, such as antioxidant [33] and antiproliferative of rat aortic smooth muscle cells [34]. Xia et al. [35] showed that luteolin-7-glucoside is capable of enhancing eNOS gene expression in human endothelial cells. On the other hand, no effects on vascular tone were described for luteolin-7-glucoside and luteolin-7-O-pentosyl-glucoside.

Our results demonstrated that ELM-induced vascular relaxation in the rat aorta is mediated by the NO signaling pathway, at least partially, through the activation of histamine H₁ receptors. Additionally, luteolin and quercetin glycosides could contribute to the extract activity. In conclusion, ELM induces an endothelium-dependent relaxation on the rat aorta, indicating that it could be considered a source of natural bioactive products with vasodilatory activity.

Materials and Methods

Plant material and extraction

Leaves of M. moricandiana were collected in February 2011 at the Parque Nacional da Restinga de Jurubatiba, Quissamã, Rio de Janeiro, Brazil (22°27′S, 41°65′W). Botanical identification was performed by Dra. Tatiana Ungaretti Paleo Konno and a voucher specimen has been deposited at the Universidade Federal do Rio de Janeiro Herbarium under the number RFA38748. Leaves were air-dried at 40 °C, ground, and then exhaustively extracted (248 g) by maceration with ethanol/H₂O (7 : 3) at room temperature. The hydroalcoholic crude extract was obtained after the removal of the solvent under reduced pressure.

HPLC-DAD analysis for flavonoids quantification

The analyses were carried out using a Shimadzu chromatograph equipped with an LC-20AT quaternary solvent pump, SPD-M20A diode array detector, CBM-20A controller, auto-injector SIL-20A, column oven CTO-20A, DGU-20A₅ degasser, and LCsolution™ software version 1.25 SP1 for the system control. The analyses were performed at 30 °C using an RP-18 reverse-phase column (5 µm, 250 mm, 4.60 mm, Supelcosil, Supelco) and the following eluents: A: H₂O adjusted to pH 3.2 by H₃PO₄ and B: CH₃CN. The gradient (v/v) was applied as follows: 0–15 % B in 0–10 min; 15–25 % B in 10–20 min; 25–50 % B in 20-40 min and 50–100 % B in 40–47 min. The flow elution was 1 mL · min⁻¹. Ten µL samples were injected after dilution of 10 mg of ELM in 1 mL of ultrapure water.

LC-ESI-MS and ion trap LC-MS/MS analysis

LC-ESI-MS analysis was performed on a modular HPLC system from Shimadzu, which consisted of a communication bus module (CBM 20A), two pumps (LC-20AD), column oven (CTO 20A) maintained at 30 °C, autosampler (SIL-20A_HT), and diode array detector (SPD-M20AV) coupled to a micrOTOFII (Bruker Daltonics) ESI-qTOF mass spectrometer. Ion trap LC-MS/MS analysis was performed on the same HPLC system described above coupled to an Amazon SL (Bruker Daltonics) ion trap mass spectrometer.

The column (Luna 5 µm C18, 250 × 4.6 mm, Phenomenex), connected in line, was used for the chromatographic analyses. The flow rate was 1.0 mL · min⁻¹ and the injection volume was 10 µL at 1 mg · mL⁻¹. The mobile phase consisted of ultrapure H₂O (A) with the pH adjusted to 3 by formic acid and acetonitrile (B) according to the following gradient elution profile: 10–15 % B in 0–10 min; 15–25 % B in 10–20 min; 25–50 % B in 20–35 min; 50–90 % B in 35–50 min; 90–100 % B in 50–55 min; 100 % B in 55–60 min; 100–10 % B in 60–65 min; and 10 % B in 65–70 min. The column eluent was split at a ratio of 7 : 3, with the larger flow going to the DAD detector and the lower one to the mass spectrometer. HPLC-MS TIC chromatograms were recorded between 50 and 1300 m/z in the positive ionization mode. The spectrum was obtained in high resolution using a capillary and end plate with 3.5 kV and 500 V, respectively. Nitrogen was used as the nebulizer (5.5 bar). The flow of the drying gas was 10 L · min⁻¹ and the drying gas temperature was 220 °C for LC-ESI-MS and 300 °C for ion trap LC-MS/MS analysis. For chromatographic analysis, ultrapure water (Millipore), HPLC-grade acetonitrile (J. T. Baker), and acetic acid (J. T. Baker) were used.

Total flavonoid content

Calibration graphs for HPLC-DAD were recorded with rutin (≥ 94 %; quercetin 3-O-rutinoside) in amounts ranging from 0.20 to 10.0 µg with ten data points. Rutin was chosen as the external standard in order to propose a feasible HPLC-DAD method for routine analyses of the total flavonoid content [36] and also due to the presence of this compound and related metabolites in the ELM extract. The linearity range of the detector response was verified using a series of twofold diluted solutions of rutin. The relationship between peak areas (detector responses) and the amount of rutin was linear over 1000–20 µg/mL (r² = 0.9999). To evaluate the repeatability of the injection integration, the rutin standard solution and the extract ELM were injected three times and the relative standard deviation values were calculated.

Preparation of aortic rings

All animal protocols were approved by the Animal Care and Use Committee at Universidade Federal do Rio de Janeiro on 14 March 2012, under the license MACAÉ01.

The thoracic aorta was dissected from male Wistar rats (200–250 g), carefully cleaned of connective tissue, and cut into 3–4 mm rings. Aortic rings were placed in vertical chambers filled with 10 mL of saline solution composed of (in mM): NaCl, 123.0; KCl, 4.7; MgCl₂, 1.2; NaH₂PO₄, 1.2; NaHCO₃, 15.5; CaCl₂, 1.2; glucose, 11.5 (pH 7.4) and oxygenated with carbogen gas at 37 ± 0.5 °C. Each aorta ring was mounted between two hooks in which one was attached to a force transducer (MLT0201; ADInstruments), whose signal was digitalized (Power Lab 4/30; ADInstruments) and stored on a computer for analysis using the software LabChart Pro (ADInstruments). After equilibrium under 1 g of resting tension for 90 min, the integrity of the endothelium was assessed by determining the relaxation response to acetylcholine (10 µM) in phenylephrine (10 µM) precontracted rings. Endothelium was considered functional if acetylcholine-induced relaxation was greater than 80 %. In some rings, the endothelium was mechanically removed by gently rubbing the luminal surface with plastic tubing. Removal of the endothelium was confirmed by the lack of relaxation in response to acetylcholine. Then, the contractile response induced by phenylephrine was measured before and after exposure of aortic rings to increasing concentrations of ELM (1–100 µg/mL). The effect of the solvent DMSO alone was also evaluated.

In order to determine the mechanisms involved in ELM-induced vasodilation, aortic rings with an intact endothelium were pretreated for 15–30 min with the NO synthase inhibitor L-NAME (100 µM), the sGC inhibitor ODQ (10 µM), the cyclooxygenase inhibitor indomethacin (10 µM), the nonselective K⁺ channel blocker TEA (100 µM), the nonselective muscarinic receptor antagonist atropine (10 µM), the histamine H₁ receptor antagonist diphenhydramine (10 µM), the nonselective estrogen receptor antagonist fulvestrant (10 µM), or the bradykinin B2 receptor antagonist HOE 140 (1 µM).

Chemicals and drugs

Phenylephrine, acetylcholine, L-NAME (≥ 98 %), indomethacin (≥ 99 %), diphenhydramine (≥ 98 %), ODQ, glibenclamide, HOE-140 (≥ 94 %), wortmannin, fulvestrant (≥ 98 %), atropine (≥ 99 %), and TEA (≥ 98 %) were purchased from Sigma Chemical Co. and were dissolved in distilled water, except for indomethacin, which was dissolved in DMSO.

Statistical analysis

Relaxation induced by ELM was expressed as percentage of maximal tension observed in the presence of phenylephrine. All data were expressed as mean ± S. E. M. and differences between groups were considered statistically significant when p < 0.05. One-way analysis of variance (ANOVA) followed by a Newman-Keuls test was used for comparison between concentration-response curves.

Supporting information

UV and ESI-MS spectra of the peaks at t_R = 18.3 min, 20.7 min, 22.6 min, and 23.3 min, and ion trap MS/MS spectra of the peaks at t_R = 20.7 min and 23.3 min are available as Supporting Information.

Acknowledgements

This work was supported by FAPERJ, CNPq and FUNEMAC. In addition, this work was supported, in part, by fellowships from FAPERJ (to L. L. D. M. F.) and FUNEMAC (to B. M. P.).

Conflict of Interest

The authors declare no conflicts of interest.

• References

• 1 Calixto JB, Yunes RA. Effect of a crude extract of Mandevilla velutina on contractions induced by bradykinin and [des-Arg⁹]-bradykinin in isolated vessels of the rabbit. Br J Pharmacol 1986; 88: 937-941
  CrossrefPubMedSearch in Google Scholar
• 2 Calixto JB, Yunes RA, Cruz AB, Medeiros YS. Effect of compounds from Mandevilla velutina on bradykinin-mediated contractile and relaxant responses of the isolated guinea pig trachea. Agents Actions 1992; 36: 222-229
  PubMedSearch in Google Scholar
• 3 Mattos WM, Campos MM, Fernandes ES, Ferreira J, Richetti GP, Niero R, Yunes RA, Calixto JB. Anti-edematogenic effects of velutinol A isolated from Mandevilla velutina: evidence for a selective inhibition of kinin B1 receptor-mediated responses. Regul Pept 2006; 136: 98-104
  CrossrefPubMedSearch in Google Scholar
• 4 Biondo R, Soares AM, Bertoni BW, França SC, Pereira AMS. Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom. Plant Cell Rep 2004; 22: 549-552
  CrossrefPubMedSearch in Google Scholar
• 5 Calixto JB, Nicolau M, Yunes RA. The selective antagonism of bradykinin action on rat isolated uterus by crude Mandevilla velutina extract. Br J Pharmacol 1985; 85: 729-731
  CrossrefPubMedSearch in Google Scholar
• 6 Niero R, Alves RV, Filho VC, Calixto JB, Hawkes JE, SantʼAna AE, Yunes RA. A new anti-oedematogenic nor-pregnane derivative isolated from Mandevilla illustris . Planta Med 2002; 68: 850-853
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Cordeiro SZ, Simas NK, Arruda RCO, Sato A. Composition of epicuticular wax layer of two species of Mandevilla (Apocynoideae, Apocynaceae) from Rio de Janeiro, Brazil. Biochem Syst Ecol 2011; 39: 198-202
  CrossrefPubMedSearch in Google Scholar
• 8 Greenhan J, Harborne JB, Williams CA. Identification of lipophilic flavones and flavonols by comparative HPLC, TLC and UV spectral analysis. Phytochem Anal 2003; 14: 100-118
  CrossrefPubMedSearch in Google Scholar
• 9 Justino GC, Borges CM, Florêncio MH. Electrospray ionization tándem mass spectrometry fragmentation of protonated flavone and flavonol aglycones: a re-examination. Rapid Commun Mass Spectrom 2009; 23: 237-248
  CrossrefPubMedSearch in Google Scholar
• 10 Morgado M, Cairrão E, Santos-Silva AJ, Verde I. Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle. Cell Mol Life Sci 2012; 69: 247-266
  CrossrefPubMedSearch in Google Scholar
• 11 Zhao Y, Vanhoutte PM, Leung SWS. Vascular nitric oxide: Beyond eNOS. J Pharmacol Sci 2015; 129: 83-94
  CrossrefPubMedSearch in Google Scholar
• 12 Parkington HC, Coleman HA, Tare M. Prostacyclin and endothelium-dependent hyperpolarization. Pharmacol Res 2004; 49: 509-514
  CrossrefPubMedSearch in Google Scholar
• 13 Achike FI, Kwan CY. Nitric oxide, human diseases and the herbal products that affect the nitric oxide signalling pathway. Clin Exp Pharmacol Physiol 2003; 30: 605-615
  CrossrefPubMedSearch in Google Scholar
• 14 Michel T, Vanhoutte PM. Cellular signaling and NO production. Pflugers Arch 2010; 459: 807-816
  CrossrefPubMedSearch in Google Scholar
• 15 Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R, Haas HL. International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol Rev 1997; 49: 253-278
  PubMedSearch in Google Scholar
• 16 Kim EY, Jho HK, Kim DI, Rhyu MR. Cirsium japonicum elicits endothelium-dependent relaxation via histamine H₁-receptor in rat thoracic aorta. J Ethnopharmacol 2008; 116: 223-227
  CrossrefPubMedSearch in Google Scholar
• 17 Nakamura Y, Matsumoto H, Todoki K. Endothelium-dependent vasorelaxation induced by black currant concentrate in rat thoracic aorta. Jpn J Pharmacol 2002; 89: 29-35
  CrossrefPubMedSearch in Google Scholar
• 18 Raimundo JM, Trindade AP, Velozo LS, Kaplan MA, Sudo RT, Zapata-Sudo G. The lignan eudesmin extracted from Piper truncatum induced vascular relaxation via activation of endothelial histamine H₁ receptors. Eur J Pharmacol 2009; 606: 150-154
  CrossrefPubMedSearch in Google Scholar
• 19 Frombaum M, Clanche SL, Bonnefont-Rousselot D, Borderie D. Antioxidant effects of resveratrol and other stilbene derivatives on oxidative stress and *NO bioavailability: Potential benefits to cardiovascular diseases. Biochimie 2012; 94: 269-276
  CrossrefPubMedSearch in Google Scholar
• 20 Chalopin M, Tesse A, Martinez MC, Rognan D, Arnal JF, Adriantsitohaina RA. Estrogen receptor alpha as a key target of red wine polyphenols action on the endothelium. PLoS One 2010; 5: e8554
  CrossrefPubMedSearch in Google Scholar
• 21 Anter E, Chen K, Shapira OM, Karas RH, Keaney Jr. JF. p38 Mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols. Circ Res 2005; 96: 1072-1078
  CrossrefPubMedSearch in Google Scholar
• 22 Wu Q, Chambliss K, Umetani M, Mineo C, Shaul PW. Non-nuclear estrogen receptor signaling in the endothelium. J Biol Chem 2011; 286: 14737-14743
  CrossrefPubMedSearch in Google Scholar
• 23 Kim KH, Young BD, Bender JR. Endothelial estrogen receptor isoforms and cardiovascular disease. Mol Cell Endocrinol 2014; 389: 65-70
  CrossrefPubMedSearch in Google Scholar
• 24 Prossnitz ER, Arterburn JB. International Union of Basic and Clinical Pharmacology. XCVII. G protein-coupled estrogen receptor and its pharmacologic modulators. Pharmacol Rev 2015; 67: 505-540
  CrossrefPubMedSearch in Google Scholar
• 25 Kubota Y, Tanaka N, Umegaki K, Takenaka H, Mizuno H, Nakamura K, Shinozuka K, Kunitomo M. Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level. Life Sci 2001; 69: 2327-2336
  CrossrefPubMedSearch in Google Scholar
• 26 Li PG, Sun L, Han X, Ling S, Gan WT, Xu JW. Quercetin induces rapid eNOS phosphorylation and vasodilation by an Akt-independent and PKA-dependent mechanism. Pharmacology 2012; 89: 220-228
  CrossrefPubMedSearch in Google Scholar
• 27 Hou X, Liu Y, Niu L, Cui L, Zhang M. Enhancement of voltage-gated K⁺ channels and depression of voltage-gated Ca²⁺ channels are involved in quercetin-induced vasorelaxation in rat coronary artery. Planta Med 2014; 80: 465-472
  Thieme ConnectPubMedSearch in Google Scholar
• 28 Pérez-Vizcaíno F, Ibarra M, Cogolludo AL, Duarte J, Zaragozá-Arnáez F, Moreno L, López-López G, Tamargo J. Endothelium-independent vasodilator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries. J Pharmacol Exp Ther 2002; 302: 66-72
  CrossrefPubMedSearch in Google Scholar
• 29 Penso J, Cordeiro KC, da Cunha CR, da Silva Castro PF, Martins DR, Lião LM, Rocha ML, de Oliveira V. Vasorelaxant activity of 7-β-O-glycosides biosynthesized from flavonoids. Eur J Pharmacol 2014; 733: 75-80
  CrossrefPubMedSearch in Google Scholar
• 30 Si H, Wyeth RP, Liu D. The flavonoid luteolin induces nitric oxide production and arterial relaxation. Eur J Nutr 2014; 53: 269-275
  CrossrefPubMedSearch in Google Scholar
• 31 Qian LB, Wang HP, Chen Y, Chen FX, Ma YY, Bruce IC, Xia Q. Luteolin reduces high glucose-mediated impairment of endothelium-dependent relaxation in rat aorta by reducing oxidative stress. Pharmacol Res 2010; 61: 281-287
  CrossrefPubMedSearch in Google Scholar
• 32 Roberts RE, Allen S, Chang AP, Henderson H, Hobson GC, Karania B, Morgan KN, Pek AS, Raghvani K, Shee CY, Shikotra J, Street E, Abbas Z, Ellis K, Heer JK, Alexander SP. Distinct mechanisms of relaxation to bioactive components from chamomile species in porcine isolated blood vessels. Toxicol Appl Pharmacol 2013; 272: 797-805
  CrossrefPubMedSearch in Google Scholar
• 33 Hassan RA, Tawfik WA, Abou-Setta LM. The flavonoid constituents of Leucaena leucocephala. Growing in Egypt, and their biological activity. Afr J Tradit Complement Altern Med 2013; 11: 67-72
  PubMedSearch in Google Scholar
• 34 Kim TJ, Kim JH, Jin YR, Yun YP. The inhibitory effect and mechanism of luteolin 7-glucoside on rat aortic vascular smooth muscle cell proliferation. Arch Pharm Res 2006; 29: 67-72
  CrossrefPubMedSearch in Google Scholar
• 35 Xia N, Bollinger L, Steinkamp-Fenske K, Förstermann U, Li H. Prunella vulgaris L. upregulates eNOS expression in human endothelial cells. Am J Chin Med 2010; 38: 599-611
  CrossrefPubMedSearch in Google Scholar
• 36 Zeraik ML, Yariwake JH. Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD. Microchem J 2010; 96: 86-91
  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Calixto JB, Yunes RA. Effect of a crude extract of Mandevilla velutina on contractions induced by bradykinin and [des-Arg⁹]-bradykinin in isolated vessels of the rabbit. Br J Pharmacol 1986; 88: 937-941
  CrossrefPubMedSearch in Google Scholar
• 2 Calixto JB, Yunes RA, Cruz AB, Medeiros YS. Effect of compounds from Mandevilla velutina on bradykinin-mediated contractile and relaxant responses of the isolated guinea pig trachea. Agents Actions 1992; 36: 222-229
  PubMedSearch in Google Scholar
• 3 Mattos WM, Campos MM, Fernandes ES, Ferreira J, Richetti GP, Niero R, Yunes RA, Calixto JB. Anti-edematogenic effects of velutinol A isolated from Mandevilla velutina: evidence for a selective inhibition of kinin B1 receptor-mediated responses. Regul Pept 2006; 136: 98-104
  CrossrefPubMedSearch in Google Scholar
• 4 Biondo R, Soares AM, Bertoni BW, França SC, Pereira AMS. Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom. Plant Cell Rep 2004; 22: 549-552
  CrossrefPubMedSearch in Google Scholar
• 5 Calixto JB, Nicolau M, Yunes RA. The selective antagonism of bradykinin action on rat isolated uterus by crude Mandevilla velutina extract. Br J Pharmacol 1985; 85: 729-731
  CrossrefPubMedSearch in Google Scholar
• 6 Niero R, Alves RV, Filho VC, Calixto JB, Hawkes JE, SantʼAna AE, Yunes RA. A new anti-oedematogenic nor-pregnane derivative isolated from Mandevilla illustris . Planta Med 2002; 68: 850-853
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Cordeiro SZ, Simas NK, Arruda RCO, Sato A. Composition of epicuticular wax layer of two species of Mandevilla (Apocynoideae, Apocynaceae) from Rio de Janeiro, Brazil. Biochem Syst Ecol 2011; 39: 198-202
  CrossrefPubMedSearch in Google Scholar
• 8 Greenhan J, Harborne JB, Williams CA. Identification of lipophilic flavones and flavonols by comparative HPLC, TLC and UV spectral analysis. Phytochem Anal 2003; 14: 100-118
  CrossrefPubMedSearch in Google Scholar
• 9 Justino GC, Borges CM, Florêncio MH. Electrospray ionization tándem mass spectrometry fragmentation of protonated flavone and flavonol aglycones: a re-examination. Rapid Commun Mass Spectrom 2009; 23: 237-248
  CrossrefPubMedSearch in Google Scholar
• 10 Morgado M, Cairrão E, Santos-Silva AJ, Verde I. Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle. Cell Mol Life Sci 2012; 69: 247-266
  CrossrefPubMedSearch in Google Scholar
• 11 Zhao Y, Vanhoutte PM, Leung SWS. Vascular nitric oxide: Beyond eNOS. J Pharmacol Sci 2015; 129: 83-94
  CrossrefPubMedSearch in Google Scholar
• 12 Parkington HC, Coleman HA, Tare M. Prostacyclin and endothelium-dependent hyperpolarization. Pharmacol Res 2004; 49: 509-514
  CrossrefPubMedSearch in Google Scholar
• 13 Achike FI, Kwan CY. Nitric oxide, human diseases and the herbal products that affect the nitric oxide signalling pathway. Clin Exp Pharmacol Physiol 2003; 30: 605-615
  CrossrefPubMedSearch in Google Scholar
• 14 Michel T, Vanhoutte PM. Cellular signaling and NO production. Pflugers Arch 2010; 459: 807-816
  CrossrefPubMedSearch in Google Scholar
• 15 Hill SJ, Ganellin CR, Timmerman H, Schwartz JC, Shankley NP, Young JM, Schunack W, Levi R, Haas HL. International Union of Pharmacology. XIII. Classification of histamine receptors. Pharmacol Rev 1997; 49: 253-278
  PubMedSearch in Google Scholar
• 16 Kim EY, Jho HK, Kim DI, Rhyu MR. Cirsium japonicum elicits endothelium-dependent relaxation via histamine H₁-receptor in rat thoracic aorta. J Ethnopharmacol 2008; 116: 223-227
  CrossrefPubMedSearch in Google Scholar
• 17 Nakamura Y, Matsumoto H, Todoki K. Endothelium-dependent vasorelaxation induced by black currant concentrate in rat thoracic aorta. Jpn J Pharmacol 2002; 89: 29-35
  CrossrefPubMedSearch in Google Scholar
• 18 Raimundo JM, Trindade AP, Velozo LS, Kaplan MA, Sudo RT, Zapata-Sudo G. The lignan eudesmin extracted from Piper truncatum induced vascular relaxation via activation of endothelial histamine H₁ receptors. Eur J Pharmacol 2009; 606: 150-154
  CrossrefPubMedSearch in Google Scholar
• 19 Frombaum M, Clanche SL, Bonnefont-Rousselot D, Borderie D. Antioxidant effects of resveratrol and other stilbene derivatives on oxidative stress and *NO bioavailability: Potential benefits to cardiovascular diseases. Biochimie 2012; 94: 269-276
  CrossrefPubMedSearch in Google Scholar
• 20 Chalopin M, Tesse A, Martinez MC, Rognan D, Arnal JF, Adriantsitohaina RA. Estrogen receptor alpha as a key target of red wine polyphenols action on the endothelium. PLoS One 2010; 5: e8554
  CrossrefPubMedSearch in Google Scholar
• 21 Anter E, Chen K, Shapira OM, Karas RH, Keaney Jr. JF. p38 Mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols. Circ Res 2005; 96: 1072-1078
  CrossrefPubMedSearch in Google Scholar
• 22 Wu Q, Chambliss K, Umetani M, Mineo C, Shaul PW. Non-nuclear estrogen receptor signaling in the endothelium. J Biol Chem 2011; 286: 14737-14743
  CrossrefPubMedSearch in Google Scholar
• 23 Kim KH, Young BD, Bender JR. Endothelial estrogen receptor isoforms and cardiovascular disease. Mol Cell Endocrinol 2014; 389: 65-70
  CrossrefPubMedSearch in Google Scholar
• 24 Prossnitz ER, Arterburn JB. International Union of Basic and Clinical Pharmacology. XCVII. G protein-coupled estrogen receptor and its pharmacologic modulators. Pharmacol Rev 2015; 67: 505-540
  CrossrefPubMedSearch in Google Scholar
• 25 Kubota Y, Tanaka N, Umegaki K, Takenaka H, Mizuno H, Nakamura K, Shinozuka K, Kunitomo M. Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level. Life Sci 2001; 69: 2327-2336
  CrossrefPubMedSearch in Google Scholar
• 26 Li PG, Sun L, Han X, Ling S, Gan WT, Xu JW. Quercetin induces rapid eNOS phosphorylation and vasodilation by an Akt-independent and PKA-dependent mechanism. Pharmacology 2012; 89: 220-228
  CrossrefPubMedSearch in Google Scholar
• 27 Hou X, Liu Y, Niu L, Cui L, Zhang M. Enhancement of voltage-gated K⁺ channels and depression of voltage-gated Ca²⁺ channels are involved in quercetin-induced vasorelaxation in rat coronary artery. Planta Med 2014; 80: 465-472
  Thieme ConnectPubMedSearch in Google Scholar
• 28 Pérez-Vizcaíno F, Ibarra M, Cogolludo AL, Duarte J, Zaragozá-Arnáez F, Moreno L, López-López G, Tamargo J. Endothelium-independent vasodilator effects of the flavonoid quercetin and its methylated metabolites in rat conductance and resistance arteries. J Pharmacol Exp Ther 2002; 302: 66-72
  CrossrefPubMedSearch in Google Scholar
• 29 Penso J, Cordeiro KC, da Cunha CR, da Silva Castro PF, Martins DR, Lião LM, Rocha ML, de Oliveira V. Vasorelaxant activity of 7-β-O-glycosides biosynthesized from flavonoids. Eur J Pharmacol 2014; 733: 75-80
  CrossrefPubMedSearch in Google Scholar
• 30 Si H, Wyeth RP, Liu D. The flavonoid luteolin induces nitric oxide production and arterial relaxation. Eur J Nutr 2014; 53: 269-275
  CrossrefPubMedSearch in Google Scholar
• 31 Qian LB, Wang HP, Chen Y, Chen FX, Ma YY, Bruce IC, Xia Q. Luteolin reduces high glucose-mediated impairment of endothelium-dependent relaxation in rat aorta by reducing oxidative stress. Pharmacol Res 2010; 61: 281-287
  CrossrefPubMedSearch in Google Scholar
• 32 Roberts RE, Allen S, Chang AP, Henderson H, Hobson GC, Karania B, Morgan KN, Pek AS, Raghvani K, Shee CY, Shikotra J, Street E, Abbas Z, Ellis K, Heer JK, Alexander SP. Distinct mechanisms of relaxation to bioactive components from chamomile species in porcine isolated blood vessels. Toxicol Appl Pharmacol 2013; 272: 797-805
  CrossrefPubMedSearch in Google Scholar
• 33 Hassan RA, Tawfik WA, Abou-Setta LM. The flavonoid constituents of Leucaena leucocephala. Growing in Egypt, and their biological activity. Afr J Tradit Complement Altern Med 2013; 11: 67-72
  PubMedSearch in Google Scholar
• 34 Kim TJ, Kim JH, Jin YR, Yun YP. The inhibitory effect and mechanism of luteolin 7-glucoside on rat aortic vascular smooth muscle cell proliferation. Arch Pharm Res 2006; 29: 67-72
  CrossrefPubMedSearch in Google Scholar
• 35 Xia N, Bollinger L, Steinkamp-Fenske K, Förstermann U, Li H. Prunella vulgaris L. upregulates eNOS expression in human endothelial cells. Am J Chin Med 2010; 38: 599-611
  CrossrefPubMedSearch in Google Scholar
• 36 Zeraik ML, Yariwake JH. Quantification of isoorientin and total flavonoids in Passiflora edulis fruit pulp by HPLC-UV/DAD. Microchem J 2010; 96: 86-91
  CrossrefPubMedSearch in Google Scholar

• Supplementary Material