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Journal of Ethnopharmacology
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a r t i c l e i n f o a b s t r a c t
Article history: Ethnopharmacological relevance: Solanum paludosum Moric. (jurubeba-roxa) is commonly used to treat
Received 2 December 2011 hypertension as a substitute for Solanum paniculatum L. (jurubeba verdadeira). The total ethanolic extract
Received in revised form 13 February 2012 from the root bark of Solanum paludosum have been found to cause hypotension in rats.
Accepted 17 March 2012
Aim of the study: To investigate the mechanism by which the total alkaloid fraction obtained from the
Available online 26 March 2012
root bark of Solanum paludosum (FAT-SP) acts as a vasorelaxant agent on rat thoracic aorta.
Materials and methods: Rings of rat aorta were suspended in organ bath containing Krebs solution at 37 ◦ C,
Keywords:
bubbled with carbogen mixture (95% O2 and 5% CO2 ) under a resting tension of 1 g. Isometric contractions
Solanum paludosum
Total alkaloids
were measured using a force transducer coupled to an amplifier and a microcomputer.
Rat aorta Results: FAT-SP has been found cause relaxation of the aortic rings pre-contracted with phenyle-
NO/sCG/PKG pathway phrine (Phe) in a concentration-dependent manner, in the presence and absence of endothelium.
Potassium channels This effect was more potent on the endothelium-intact aorta. In the presence of endothelium, nei-
ther indomethacin (non-selective cyclooxygenase inhibitor) nor atropine (non-selective muscarinic
receptor antagonist), produced significant changes on the relaxation response. On the other hand,
in the presence of calmidazolium (a calmodulin inhibitor), N-nitro-l-arginine methyl ester (l-NAME,
nitric oxide synthase inhibitor), hydroxocobalamin (HDX) (scavenger of free-radical nitric oxide), 1-
H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, selective blocker of soluble guanylate cyclase),
Rp-8-bromo--phenyl-1,N2 -ethenoguanosine 3 :5 -cyclic monophosphorothioate sodium salt hydrate
(Rp-8-Br-PET-cGMPS, competitive inhibitor of cGMP-dependent protein kinase G) or TEA+ (tetraethy-
lammonium, nonselective potassium channel blocker), the vasorelaxant effect was significantly reduced,
suggesting the involvement of NO/sCG/PKG pathway and potassium channel opening in vasorelaxant
action of the FAT-SP.
Conclusion: The mechanism of vasorelaxant activity of the FAT-SP on rat aorta involves both NO/sCG/PKG
pathway and potassium channels.
© 2012 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2012.03.032
896 F.S. Monteiro et al. / Journal of Ethnopharmacology 141 (2012) 895–900
endothelial dysfunction. Hypertension is one of the most important Tetraethylammonium chloride (TEA+ ), dimethyl sulfoxide
public health problems in Brazil (Longo et al., 2009). (DMSO), histamine hydrochloride, atropine sulfate, indomethacin,
Antihypertensive drugs lower the blood pressure by acting on NG -nitro-l-arginine methyl ester (l-NAME), phenylephrine
the resistance vessels, the capacitance vessels, the heart, and the (Phe), 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ),
kidney. Diuretics, sympatholytic drugs, vasodilators, calcium chan- Rp-8-bromo--phenyl-1,N2 -ethenoguanosine 3 :5 -cyclic
nel blockers, angiotensin-II blockers and angiotensin converting monophosphorothioate sodium salt hydrate (Rp-8-Br-PET-cGMPS)
enzyme inhibitors are important classes of antihypertensive drugs and calmidazolium chloride were obtained from Sigma–Aldrich
(Webb et al., 2010). (USA).
Nitric oxide (NO) has been found to be, in recent years, an impor- Glucose (C6 H12 O6 ) and monobasic potassium phosphate
tant regulator of vascular functions by controlling blood vessel tone (KH2 PO4 ·H2 O) were obtained from Nuclear (Brazil).
as well as the interaction of blood cells with the vessel wall. The Sodium chloride (NaCl) and acetylcholine hydrochloride (ACh)
NO/cGMP/PKG pathway and potassium channels play an essential were obtained from Merck (Brazil).
role in vascular smooth muscle relaxation, and the clinical studies Hydroxocobalamin hydrochloride (HDX) was obtained from
indicate that endothelium-derived NO is involved in normal and Bristol-Myers Squibb (Equador). Carbogen mixture (95% O2 and 5%
pathological blood pressure regulation in humans (Moncada and CO2 ) was obtained from White Martins (Brazil).
Higgs, 2006). FAT-SP, provided by our collaborator of phytochemistry
Solanum paludosum is a shrub commonly known as “jurubeba- (Bhattacharyya et al., 2009) was dissolved in distilled water to the
roxa” in northeastern Brazil (Agra and Bhattacharyya, 1999), and concentration of (10 mg/mL) and stored at 0 ◦ C. The stock solu-
the root bark of this plant is often used in folk medicine to tion was diluted in distilled water according to each experimental
treat hypertension and other diseases as a substitute for Solanum protocol.
paniculatum (jurubeba-verdadeira) (Ribeiro et al., 1986). All substances were kept in freezer at −20 ◦ C except HDX which
In a recent study aimed to investigate the pharmacological was kept under room temperature. Indomethacin was dissolved
properties, the total alkaloid fraction of the root bark of Solanum and diluted in a in a 0.2 M NaHCO3 solution and ODQ in DMSO.
paludosum (FAT-SP) did not show any hemolytic activity on ery-
throcytes of rats and displayed spasmolytic activity on rat uterus 2.4. Animals
and guinea-pig trachea (Correia et al., 2011).
Male Wistar rats (Rattus norvegicus), 12 weeks old and weighing
A chemical study of the FAT-SP resulted in the identification
250–300 g each, obtained from Bioterium Prof. Thomas George of
(IR, 1 H and 13 C NMR with APT and DEPT) of the following com-
UFPB, were used in this study. The animals were housed in cages
pounds: N-hydroxysolasodine, leptinidine, tomatidenol and a new
at a temperature of 21 ± 1 ◦ C in a 12 h light/12 h dark cycle and free
compound, putuline (see Bhattacharyya et al., 2009).
access to food and water. All procedures were approved by the UFPB
Moreover, the total ethanolic extract of the root bark of Solanum
Animal Research Ethics Committee (protocol/CEPA no. 0111/09).
paludosum has been found to have curare-like and hypotensive
activity in rats (Ataíde, “unpublished results”). Therefore, we inves-
2.5. Tissue preparation
tigated the vasorelaxant effect and action mechanism of the FAT-SP
on isolated rat aorta. All rats were euthanized by decapitation with guillotine. The
aortic rings about 3–5 mm wide were obtained from the thoracic
2. Methods aorta and were freed from the surrounding connective tissue. To
obtain isometric responses, the rings were individually suspended
2.1. Plant material on stainless steel rods in organ baths (6 mL) containing normal
Krebs solution at 37 ◦ C with the following composition (mM): NaCl
The plant material of Solanum paludosum was collected in (118.0), NaHCO3 (25.0), KCl (4.6), MgSO4 (5.7), KH2 PO4 (1.1), CaCl2
November 2005 in the municipality of Santa Rita, State of Paraíba, (2.5) and glucose (11.0). The test solutions were stabilized for 1 h.
Brazil. The material was collected and identified by Maria de Fátima During this period, the aortic rings were kept under a resting ten-
Agra, and the voucher specimen (Agra 6734) is deposited at the sion of 1 g. In the meantime, the nutrient solution was renewed
Prof. Lauro Pires Xavier (JPB) Herbarium, Universidade Federal da every 15 min to prevent metabolite interference (Altura and Altura,
Paraíba, João Pessoa, PB, Brazil. 1970). In some of the rings, usual care was taken to avoid abrasion
of the inner surface and maintain the integrity of the endothelial
layer. In the remaining aortic rings, endothelial cells were removed
2.2. Extraction of FAT-SP
by gently rubbing the inner surface with moistened cotton swabs.
After the initial stabilization period, a contraction was induced
The dried and powdered root bark (1.1 kg) of Solanum paludo-
with Phe (0.3 M) and when the tonic phase was reached
sum was extracted with MeOH:H2 O (95:5) until the last extract
(12–15 min), ACh (1 M) was added to all preparations to verify
was free from color. The solvent was then removed under reduced
endothelium integrity (Furchgott and Zawadzki, 1980). The vas-
pressure, 5% H2 SO4 was added and the mixture was left for stand-
cular endothelium was considered intact when the aortic rings
ing overnight. The product was then filtered through celite and
showed more than 50% of relaxation (Ajaya et al., 2003). Endothe-
the filtrate was extracted with ether to remove fatty material. The
lium removal was confirmed by the absence of relaxation after ACh
aqueous acid phase was then cooled, basified with NH4 OH to pH 9
was added to the bath, or when the relaxation was lower than 10%,
and left standing for a few hours. The precipitated alkaloids were
when these rings were considered free of functional endothelium
collected by filtration which is designated FAT-SP (Scheme in Sup-
(Fig. 1).
plementary Material).
2.6. Experimental protocol
2.3. Drugs
2.6.1. Effect of FAT-SP on Phe-induced tonic contractions in
Magnesium sulfate heptahydrate (MgSO4 ·7H2 O), calcium chlo- endothelium-intact and -denuded rat aortic rings
ride dihydrate (CaCl2 ·2H2 O), potassium chloride (KCl) and sodium Aortic rings were obtained as described in Section 2.5. After
bicarbonate (NaHCO3 ) were obtained from Vetec (Brazil). the verification of endothelium integrity and during the tonic
F.S. Monteiro et al. / Journal of Ethnopharmacology 141 (2012) 895–900 897
Fig. 1. Representative originals records in the absence (control, A) and presence of FAT-SP on endothelium-intact (B) and denuded (C) rat aorta rings. Arrow represents
concentration of FAT-SP (1, 3, 9, 27, 81, 243, 500 and 750 g/mL). Phe: phenilefrine and W: washout.
component of a second response to the agonist, FAT-SP was cumu- selective soluble guanylyl cyclase (sCG) blocker (10 M for 15 min)
latively added to the bath in different preparations. The relaxation (Garthwaite et al., 1995); and Rp-8-Br-PET-cGMPS, a competi-
was expressed as the reversal percentage of initial contraction tive cGMP-dependent protein kinase G (PKG) inhibitor (30 M for
elicited by contractile agents and EC50 values (concentration of a 30 min) (Ibarra-Alvarado et al., 2002).
substance that produces 50% of its maximum effect) were obtained In the tonic component of the second Phe-induced contraction
with nonlinear regression from FAT-SP concentration–response in the presence of each blocker, FAT-SP was cumulatively added to
curves of rat aortic rings with both endothelium-intact and - the bath in different preparations. The relaxation was expressed as
denuded aorta. previously described.
2.6.2. Effect of FAT-SP on Phe-induced tonic contractions in 2.6.4. Effect of FAT-SP on KCl-induced (30 or 80 mM) tonic
endothelium-intact rat aortic rings in the presence of atropine or contractions in endothelium-intact rat aortic rings
indomethacin The aortic rings were obtained as described in Section 2.5. After
The aorta was mounted as described in Section 2.5. Before washing the preparation and during the tonic component of a
taking the second concentration–response curve induced by Phe, second response achieved with KCl (30 or 80 mM) solution (Pérez-
the preparations with endothelium were separately incubated for Vizcaíno et al., 1998), FAT-SP was added cumulatively to the bath, in
15 min with atropine (1 M), a nonselective muscarinic receptor different preparations. The relaxation was expressed as previously
antagonist (Barnes, 2000) or indomethacin (10 M), a nonselective described.
inhibitor of cyclooxygenase (Sigthorsson et al., 2000). Even in the
presence of blockers, an additional Phe contraction was obtained. 2.6.5. Effect of FAT-SP on Phe-induced tonic contractions in
Subsequently, FAT-SP was cumulatively added to the organ bath endothelium-intact rat aortic rings in the presence of TEA+
and on the sustained component of contraction. The relaxation The aorta was obtained as described in Section 2.5. Before
was expressed as a percentage of the reverse contraction produced inducing the second Phe-induced contraction in rings with intact
by Phe. EC50 values were calculated by the nonlinear regression endothelium, TEA+ (10 mM), a nonselective potassium channel
method from the relaxation curves obtained both in the presence blocker (Sordi et al., 2011), was added in different preparations.
and in the absence of blockers. In the tonic component of the second Phe-induced contraction in
the presence of TEA+ , FAT-SP was cumulatively added to the bath in
2.6.3. Effect of FAT-SP on Phe-induced tonic contractions in different preparations. The relaxation was expressed as previously
endothelium-intact rat aortic rings in the presence of described.
calmidazolium, l-NAME, HDX, ODQ or Rp-8-Br-PET-cGMPS
The aorta was obtained as described in Section 2.5. Before 2.7. Statistical analysis
inducing the second Phe contraction in rings with functional
endothelium, the following agents were added in different prepa- The values were expressed as mean ± S.E.M. Statistical analysis
rations: l-NAME, a competitive NO synthase inhibitor (100 M for was performed using Graph-Pad Prism 5.01 software (Graph-
30 min) (Rees et al., 1990); calmidazolium, a calmodulin inhibitor Pad Software Inc., San Diego, CA, USA). The differences between
(10 M for 30 min) (Schini and Vanhoutte, 1992), HDX, a NO rad- mean values were statistically compared using t-test and one-way
ical scavenger (30 M for 30 min) (Broderick et al., 2005); ODQ, a ANOVA, followed by Bonferroni correction when applicable, and
898 F.S. Monteiro et al. / Journal of Ethnopharmacology 141 (2012) 895–900
0 0
25 25
Relaxation (%)
Relaxation (%)
50 50
Phe
75 Endothelium-intact 75 Indomethacin
Endothelium-denuded
Atropine
100 100
0 1 2 3 0 1 2 3
log [FAT-SP] µg/mL log [FAT-SP] µg/mL
Fig. 2. Effect of FAT-SP on Phe-induced (0.3 M) tonic contraction in rat aorta rings Fig. 3. Effect of FAT-SP on Phe-induced (0.3 M) tonic contraction in endothelium-
with intact (䊉) or denuded () endothelium. Symbols and vertical bars represent intact rat aorta rings in absence (control, 䊉) and presence of atropine (1 M, )
means and S.E.M., t-test, ***p < 0.001 (endothelium-intact vs. endothelium-denuded) or indomethacin (10 M, ). Symbols and vertical bars represent means and S.E.M
(n = 5). (n = 5).
were considered to differ significantly when p < 0.05. The EC50 val- calmidazolium, a calmodulin inhibitor to investigate a possible
ues were determined using nonlinear regression (Neubig et al., calmodulin role in the relaxation produced by FAT-SP on rat aorta.
2003). Since this effect produced by FAT-SP was attenuated with the
decrease in the relaxation potency (EC50 = 379.0 ± 64.8 g/mL) and
3. Results and discussion maximum effect reduction (Fig. 4) in the presence of calmida-
zolium, it appears that vasorelaxant effect of the FAT-SP involves
This study demonstrated for the first time that the total alka- calmodulin participation.
loid fraction obtained from the root bark of Solanum paludosum The endothelium-derived NO is more likely free radical (NO• )
(FAT-SP) shows vasorelaxant effect in isolated rat aorta. Moreover, form (Feelisch et al., 1994). Therefore, we used HDX, a NO•
the most significant finding concerns the characterization of the scavenger, to investigate NO role in the relaxation produced
vasorelaxant action mechanism, which includes the activation of by FAT-SP on rat aorta. The relaxation potency and effect
NO/sCG/PKG pathway and the potassium channels. The presence (EC50 = 294.9 ± 20.4 g/mL) of FAT-SP was reduced (Fig. 4) in the
of steroid alkaloids may be responsible for the vasorelaxant effect presence of HDX, it is suggestive that vasorelaxant effect of the
of FAT-SP. FAT-SP involves NO participation.
The FAT-SP relaxed rat aorta pre-contracted with Phe Solanum species are known to be rich in steroid alkaloids and
(0.3 M) in a concentration-dependent manner on both intact several species present relevant hypotensive activities. It has been
(EC50 = 75.4 ± 6.2 g/mL) and denuded (EC50 = 242.8 ± 11.7 g/mL) reported in the literature that substances with a steroidal skeleton
endothelium (Fig. 2). The fact that vasorelaxant effect of the FAT-SP
was more potent on endothelium-intact rat aorta is suggestive of
500
the involvement of endothelium-derived relaxing factors (EDRF)
such as NO and prostacyclin (PGI2 ). The Phe-induced contraction
**
400
was reversible 30 min after the withdrawal of FAT-SP from the bath ***
EC50 (µg/mL)
Q
ol
PS
E
M
H
O
on
ol
cG
az
C
L-
T-
id
r-
B
8-
0 0
25 25
Relaxation (%)
Relaxantion (%)
50 50
KCl 30 mM Phe
75 75
KCl 80 mM TEA+
100 100
0 1 2 3 0 1 2 3
log [FAT-SP] µg/mL log [FAT-SP] µg/mL
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