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Endothelium-dependent vasorelaxation in rat thoracic aorta by Mansoa hirsuta D.C.


The vasodilator effect of the ethanolic extract of Mansoa hirsuta leaves (EEF) was assayed in rat aortic rings. EEF produced a concentration-dependent vasodilatation ([pIC.sub.50] = 5.1[+ or -]0.2), which was absent in endothelium-denuded vessels. The vasodilator effect of EEF was similar to a standardized ethanolic extract of Hancornia speciosa Gomes ([pIC.sub.50] = 5.1 [+ or -] 0.1). The endothelium-dependent vasodilatation induced by EEF was abolished by L-NAME (100 [mu]M), a nitric oxide (NO) synthase inhibitor, but not by indomethacin (10[mu]M; [pIC.sub.50] = 4.9 [+ or -] 0.2), a cyclooxygenase inhibitor. The concentration-response curve of EEF was not modified by the addition of superoxide dismutase (SOD; 300 U/ml). In addition, EEF (50 [mu]g/ml) displaced the 3-morpholino-sidnonimine (SIN-1; p<0.05) concentration-effect curve to the left, as well as SOD (300 U/ml). These findings lead us to conclude that EEF induces a NO- and endothelium-dependent vasodilatation in rat aortic preparations, and that this effect is, at least in some extent, due to an increase in the NO bioavailability as consequence of its antioxidant activity. The HPLC-DAD profile recorded for EEF indicates the presence of four major peaks with close retention times, exhibiting similar UV spectra with wavelength maxima compatible with heterogeneous proanthocyanidins.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Mansoa hirsuta; Bignoniaceae; Vasorelaxation; Nitric oxide; Antioxidant; Rat aortic rings; Proanthocyanidins


Mansoa hirsute D.C. (Bignoniaceae) is a liana found in the Brazilian Atlantic forest. Secondary metabolites belonging to different classes have already been reported for Bignoniaceae species, including naphthoquinones (Morais et al., 2007; Lenta et al., 2007), lignans (Kanchanapoom et al., 2006), triterpenes (Leite et al., 2006) and flavonoids (Pauletti et al., 2003). Chemical data are scarce for Mansoa, being restricted to two anti-tumor naphthoquinones reported for M. alliaceae (Itokawa et al., 1991) and to alkanols identified by GC-MS in M. hirsuta leaves (Rocha et al., 2004), along with ursolic acid, lupeol, [beta]-sitosterol and stigmasterol isolated from the leaves of the same species (Silva et al., 2006).

As part of a study directed towards the screening of Brazilian plants with potential anti-hypertensive activity, we have previously reported the in vitro angiotensin-converting enzyme (ACE) inhibition of an ethanol extract from M. hirsuta leaves (Braga et al., 2000). This finding drove us to investigate the vascular effects of M. hirsuta, since other ACE-inhibiting plants, such as Ouratea semiserrata and Hancornia speciosa, also exhibited strong vasodilator activity (Braga et al., 2000; Cortes et al., 2002; Ferreira et al., 2007a, b).

Materials and methods

Plant material

The leaves of Mansoa hirsuta D.C. were collected in Caratinga, Minas Gerais state, Brazil, in 1998. The species was identified by Dr. J. A. Lombardi, from the Botanical Department, Instituto de Ciencias Biologicas, UFMG, Belo Horizonte, Brazil, where a voucher sample (BHCB 23.862) is deposited.

Extract (EEF) preparation

After drying at 40 [degrees]C for 72 h, the plant material was powdered and extracted with 96% EtOH by exhaustive percolation. The solvent was vacuum removed in a rotatory evaporator at 50 [degrees]C, leaving a dark residue (EEF), which was kept in a desiccator until constant weight. The drug to extract ratio was ca. 3.5:1.

HPLC characterization of EEF

Analyses were carried out on a Waters alliance 2695 HPLC system composed of a quaternary pump, an auto sampler, a photodiode array detector (DAD) 2996 and a Waters Empower pro data handling system (Waters Corporation, Milford, USA). An ODS column (250 x 4.0 mm i.d., 5[mu]m; Merck, Darmstadt, Germany) was employed for the analysis. The profiles were performed employing a linear gradient of [H.sub.2]O (A) and [CH.sub.3]CN(B), 0 min 95% A, 5% B; 60min 5% A, 95% B; followed by 10 min of isocratic elution, at a temperature of 40[degrees]C and flow rate of l.0 ml/min. The chromatograms were obtained at 210 nm and UV spectra from 190 to 400 nm were recorded on line. EEF and proanthocyanidin B2, employed as reference compound, were dissolved in MeOH to concentrations of 5 and 1 nig/ml, respectively. After centrifugation at 10,000 rpm, the sample solutions (10 [mu]l) were injected onto the apparatus.


Male Wistar rats (200-250 g) from the Animal Care Facilities (CEBIO) of Instituto de Ciencias Biologieas, UFMG, were used. They were kept at 22-25 [degrees]C in a 12 h light/dark cycle, and had free access to food and water. Animal experiments were performed according to the recommendations of the Brazilian Council for Animal Care.

Rat aortic rings preparation and mounting

The descending thoracic aorta were prepared and mounted as previously described (Cruz et al., 2006).

Vasorelaxant activity in pre-contracted rat aortic rings

The determination of vasorelaxant activity was performed in aortic rings with or without functional endothelium, pre-contracted to the same tension with phenylephrine (0.1 [mu]mol/1). EEF was added in increasing cumulative concentrations once the response to phenylephrine had stabilized. To evaluate the participation of endothelium-derived products in the relaxant effect of EEF, experiments were carried out in the presence of L-NAME (100[mu]M), indomethacin (10[mu]M) or superoxide dismutase (SOD, 300 U/ml), added to the bath 20min prior to the addition of phenylephrine. The concentration-response curves to 3-morpholinosidnoni-mine (SIN-1) were obtained in endothelium-denuded rat aortic rings, in the presence and absence of EEF or SOD.

A standardized ethanolic extract of Hancornia speciosa Gomes was used as a positive control for the effect of EEF. It was obtained and tested as described by Ferreira et al. (2007a, b).


Acetylcholine chloride, indomethacin, L-NAME, L-phenylephrine chloride, SIN-1 and SOD were purchased from Sigma (St. Louis, MO, USA). Indomethacin was dissolved in 0.5% (w/v) sodium bicarbonate solution and the other drugs were dissolved in distilled water at a concentration of 10 mM. All subsequent dilutions were made immediately before use with Krebs-Henseleit solution with the following composition (mM): NaCl 135.0, KCl 5.0, [NaHCO.sub.3] 20.0, [MgSO.sub.4] 1.4, [CaCl.sub.2] 2.5, [KH.sub.2][PO.sub.4] 1.17 and glucose 11,0. Proanthocyanidin B2 was obtained from Extrasynthese (Genay Cedex, France).

Statistical analysis

The experimental data are expressed as mean [+ or -] standard error mean (S.E.M.) of at least five experiments. Statistical analyses were performed using unpaired Student's t-test. Concentration-response curves were compared by two-way ANOVA plus Bonferroni's post-test. Results were considered significantly different when p <0.05. The values of [pIC.sub.50] represent the -log of the concentration values for EEF (g/ml) and SIN-1 (mol/1) that induce 50% reduction of the sustained contraction induced by phenylephrine.

Results and discussion

EEF elicited a concentration-dependent relaxation in aortic rings with functional endothelium (Fig. 1 A), with [pIC.sub.50] of 5.1 [+ or -] 0.2. This effect was completely abolished in the absence of functional endothelium (Fig. 1A), indicating that the vasodilator effect of EEF is dependent on endothelium-derived relaxing factors. The endothelium- and NO-dependent effect of EEF is equipotent to the vasodilator effect of a standardized ethanolic extract of Hancornia speciosa Gomes ([pIC.sub.50] = 5.1 [+ or -]0.1; data not shown), previously described by us in rat aorta (Fcrreira et ah, 2007a).


To evaluate the participation of NO in the vasorelaxant activity, aortic rings were treated with L-NAME, a classical NO synthase inhibitor. In this experimental condition, the EEF-induced vasodilatation was completely abolished (Fig. IB), as similarly observed in endothelium-denuded aortic rings, suggesting that NO is the main endothelium-derived factor involved in EEF activity. On the other hand, vasodilatation was not significantly modified in aortic rings treated with indomethacin (Fig. 1B, [pIC.sub.50] = 4.9 [+ or -] 0.2), a cycloox-ygenase inhibitor, at a concentration which inhibited contraction by arachidonic acid (data not shown). This finding demonstrates that prostanoids are probably not involved in vasodilatation induced by EEF.

Endothelium-derived NO plays an important role in the control of vascular homeostasis. NO modulates the vascular tone, the growth of vascular smooth muscle cells, and decreases platelet adhesion and aggregation. It also decreases the adherence of other blood components (Moncada et al., 1991; Scott-Burden and Vanhoutte, 1994). A decrease in NO production or bioavailability is closely associated with endothelial dysfunction or injury, which is an important factor in pathologies such as atherosclerosis, restenosis and hypertension (Landmesser and Drexler, 2007).

The HPLC profile of EEF showed four major peaks with close retention times (17.24, 18.27, 19.49 and 20.70 min). The UV spectra recorded on line for these peaks indicated similar maximum wavelength absorption around 280 nm, with a shoulder approximately at 300 nm, these being values compatible with heterogeneous proanthocyanidins (Svedstrom et al., 2006; Ismailov et al., 1998). Proanthocyanidin B2 was also analyzed as reference compound, employing the same chromatographic conditions (Fig. 2B). This compound presents a [[lambda].sub.max] of 279.3 nm, close to the constituents of EEF, but showed no shoulder around 300 nm. Such difference relies on the fact that proantohcyanidin B2 is a homogeneous dimmer, whereas EEF constituents seem to be heterogeneous proanthocyanidins. TLC phytochemical screening carried out for EEF also indicated the presence of polyphenols and tannins (data not shown).


The protective effects of polyphenols on the cardiovascular system have been previously reported, including antithrombotic, antioxidant, anti-ischaemic and vasorelaxant properties, the majority of these effects being related to the antioxidant activity (for review, see Stoclet et al., 2004). The endothelium-dependent vasodilatation is greatly impaired by the increase in reactive oxygen species (ROS) production, since ROS reduce the bioavailability of NO on the vessel wall (Taniyama and Griendling, 2003; Tepel, 2003; Farre and Casado, 2001).

To investigate the antioxidant activity of EEF, the vessels were pre-treated with superoxide dismutase (SOD) and no modification in the concentration-response curve of EEF was observed (Fig. 3A). This finding suggests that ROS are not formed in significant amounts in the presence of EEF, which supports its antioxidant activity. Furthermore, the concentration response curve for morpholinosidnonimine (SIN-1), a NO donor, was shifted to the left ([pEC sub 50] = 5.6 [+ or -] 0.2 and 6.3 [+ or -] 0.1, in the absence and in presence of EEF, respectively) in the presence of EEF (50 [mu]g/ml), as illustrated in Fig. 3B. This effect was equivalent to that observed with the addition of SOD (Fig. 3B; [pEC sub 50] = 5.6 [+ or -] 0.2 and 6.1 [+ or -] 0.2, in the absence and in the presence of SOD, respectively), using a method previously described as a valuable pharmacological protocol for the characterization of antioxidant substances in isolated arteries (Andriantsitohaina et al., 1999).


It might be further mentioned that these findings are equivalent to those reported for Cuphea carthagenensis, a plant species used in the Brazilian folk medicine as antihypertensive and to treat cardiovascular diseases. The hydroalcoholic and butanolic extracts from C. carthagenensis leaves potentiate the relaxation triggered by two NO donors, S-nitroso-N-acetylpenicillamine (SNAP) and SIN-1, being phenolic compounds postulated as responsible for this effect (Schuldt et al., 2000).

Reduction on NO bioavailability, mostly due to an increase in ROS production, is closely associated with endothelial dysfunction or injury (Schulz et al., 2008). Superoxide radical ([O.sub.2 sup *-]) can react with NO at a high constant rate, generating the oxidant peroxynitrite ([ONOO sup *-]), which has stronger oxidant effect than [O.sub.2 sup *-] or NO in physiologic pH. [ONOO sup *-] has been implicated in the physiopathology of a variety of diseases, including inflammation, atherosclerosis, arthritis, endotoxemia and acute respiratory distress syndrome (Barton et al., 2001; Dhalla et al., 2000; Ischiropoulos, 1998).

Based on these reports, we can assume that the antioxidant properties of phenolics found in EEF, probably heterogeneous proanthocyanidins, may increase the half-life of NO (Stoclet et al., 2004) and therefore contribute to the relaxing effect observed in our experiments.

In conclusion, the present work demonstrates the endothelium- and NO-dependent vasodilator effect of Mansoa hirsuta ethanolic extract and suggests its relationship with the presence of proanthocyanidins.


The authors thank CNPq/Brazil (Conselho National de Desenvolvimento Cientifico e Tccnologico), FAPE-MIG/Brazil (Fundacao de Amparo a Pesquisa de Minas Gerais) and CAPES/Brazil (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior) for the financial support, graduate (PRVC) and research fellowships (FCB).


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Priscilla R.V. Campana (a), Fernao C. Braga (a), Steyner F. Cortes (b), *

(a) Faculdade de Farmacia, Unversidade Federal de Minas Gerais, Belo Horizonte, Brazil

(b) Departamento de Farmacologia, Instituto de Ciencias Biologicas. Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, CEP 31270-901, Brazil

* Corresponding author. Tel.: +5531 3409 2726; fax: +55 31 3409 2695.

E-mail address: (S.F. Cortes).

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doi: 10.1016/j.phymed.2008.09.007
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Author:Campana, Priscilla R.V.; Braga, Fernao C.; Cortes, Steyner F.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:3BRAZ
Date:May 1, 2009
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