Anti-proliferative effect of Euphorbia stenoclada in human airway smooth muscle cells in culture.

The ethanolic extract of a Malagasy species Euphorbia stenoclada (ES) (Euphorbiaceae), traditionally used as a herbal remedy against asthma and acute bronchitis, was tested to evaluate possible anti-proliferative activity on human airway smooth muscle cells (HASMC). The ES ethanolic extract totally abolished the interleukin-1beta (IL-1beta) induced proliferation of HASMC (IC(50)=0.73+/-0.08 microg/mL). No cytotoxic effect was observed up to 20 microg/mL. A bioassay-guided fractionation of the ethanolic extract was performed by reversed-phase (RP) flash chromatography, giving five fractions (FA to FE) where fraction FE was the only active one (IC(50)=0.38+/-0.02 microg/mL). The purification of this bioactive fraction FE was carried out by RP-HPLC affording six sub-fractions 1-6, and only sub-fraction 5 kept the anti-proliferative activity. Its major constituent was identified as quercetin (IC(50)=0.49+/-0.12 microg/mL) by means of HPLC/UV/MS and co-elution with the authentic standard. Quercitrin was also identified in the fraction FE but was inactive. A structure-activity relationship with flavonols determined that methylation reduced the anti-proliferative activity whereas glycosylation abolished it. The present study shows that the anti-proliferative properties of Euphorbia stenoclada are mediated through the presence of quercetin that may explain the traditional use of this plant as a remedy against asthma.

Bronchodilator activity of Phymatodes scolopendria (Burm.) Ching and its bioactive constituent.

Phymatodes scolopendria (Burm.) Ching (Polypodiaceae) is widely used in the Eastern coast of Madagascar to treat respiratory disorders. Bioassay-guided fractionation using guinea pig trachea pre-contracted with histamine to monitor the activity led to the isolation of 1,2-benzopyrone (coumarin) as the main active constituent. Effectively, it induced a concentration-dependent relaxation of the histamine with a median effective concentration (EC(50)) of 35.03 microg/ml, or carbachol (EC(50) = 33.41 microg/ml) pre-contracted guinea pig trachea, and also provoked 100% relaxation at 72.10 microg/ml. It was less active either on KCl pre-contracted trachea (EC(50) = 130.78 microg/ml) or endothelium denuded trachea (153.4 +/- 22 microg/ml). It inhibited, in a non-competitive manner, the histamine and the external calcium spasm effect on the isolated trachea but it did not significantly modify the broncho-constrictive activity of KCl. When combined with theophylline, coumarin produced a significant additive relaxing effect on pre-contracted trachea. Furthermore, its bronchodilator effect was not blocked by propranolol. In vivo, pre-treated guinea pig with coumarin showed significant resistance to histamine inhalation, with an adequate dose protecting 50% of the tested animals (AD(50)) of 75 mg/kg. These results indicate that the bronchodilator effect of coumarin is partly due to the endothelium-dependent tracheal relaxation, and may be mediated through a non-specific tracheal relaxation.

Differences between endothelin receptors mediating contraction of guinea-pig aorta and pig coronary artery.

Endothelin receptors mediating contraction were characterized and compared in rings from guinea-pig thoracic aorta and pig left circumflex coronary artery. In guinea-pig aorta, the following rank order of agonist potencies was found (mean EC50 value, nM): endothelin-1 (5.0) = endothelin-2 (5.5) > vasoactive intestinal contractor (VIC; 11.0) > sarafotoxin S6b (39.8) > [Ala3,11]endothelin-1 (121) > sarafotoxin S6a (> 150) > endothelin-3 (> 500). [Ala1,3,11,15] Endothelin-1, endothelin-(16-21), sarafotoxin S6c and sarafotoxin S6d were neither agonists nor antagonists at concentrations up to 1, 10, 3 and 1 microM, respectively. Cyclo-(D-Trp-D-Asp-Pro-D-Val-Leu) (BQ-123; 0.1-1 microM) behaved as a competitive antagonist of endothelin-1 (pA2 7.4 +/- 0.1, slope factor 0.91 +/- 0.17, n = 4). In pig coronary artery, all endothelins and sarafotoxins were agonists, except for endothelin-(16-21). Sarafotoxin S6c, [Lys4]sarafotoxin S6c, [Nle6]sarafotoxin S6c and [Ala1,3,11,15]endothelin-1 acted as partial agonists (Emax about 40% of that of endothelin-1). The rank order of agonist potencies was: sarafotoxin S6c (1.5) = [Lys4]sarafotoxin S6c (1.5) > [Nle6]sarafotoxin S6c (6.7) > or = sarafotoxin S6a (7.5) > or = endothelin-1 (12.6) > or = sarafotoxin S6b (14.8) > or = VIC (18.3) = endothelin-2 (19.3) > or = [Ala1,3,11,15]endothelin-1 (41.7) > or = [Ala3,11]endothelin-1 (55.2) > endothelin-3 (96.8) > sarafotoxin S6d (> 200). Endothelin-(16-21) was neither agonist nor antagonist at 10 microM. The concentration-response curves of endothelin-3 and sarafotoxin S6a were biphasic, consisting of a higher sensitivity (40-45% of the total effect) and a lower sensitivity component. BQ-123 (0.1-1 microM) did not alter the concentration-response curve of endothelin-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative effects of the two endothelin ETA receptor antagonists, BQ-123 and FR139317, on endothelin-1-induced contraction in guinea-pig iliac artery.

The effects of two recently introduced endothelin ETA receptor antagonists, BQ-123 and FR139317, were investigated and compared in guinea-pig isolated iliac artery. Endothelins and sarafotoxins induced contraction of guinea-pig iliac artery with a pharmacological profile characteristic of the ETA receptor. The rank order of agonist potency was (mean EC50 values, nM): endothelin-1 (11.7) > or = endothelin-2 (14.9) > or = vasoactive intestinal contractor (19.5) > sarafotoxin S6b (49.8) > or = [Ala3,11]endothelin-1 (55.0) > sarafotoxin S6a (> 100) > endothelin-3 (> or = 1000). The C-terminal hexapeptide, endothelin-(16-21), sarafotoxin S6c and sarafotoxin S6d were neither agonists nor antagonists at concentrations up to 10, 3 and 1 microM, respectively. Both FR139317 (1-10 microM) and BQ-123 (0.1-1 microM) surmountably antagonized the effects of endothelin-1. Schild analysis suggested competitive antagonism for FR139317 (Schild slope 1.32 +/- 0.21, pA2 5.82 +/- 0.16, n = 5), but not for BQ-123 (Schild slope 0.28 +/- 0.08, n = 5), which was however more potent (apparent pKB 6.6-7.2) than FR139317. The potency of FR139317 was particularly low with respect to the reported affinity for ETA receptors, suggesting heterogeneity among ETA receptors. Thus, the endothelin receptor present in guinea-pig iliac artery has the following features: (1) rank order of agonist potencies of the ETA type; (2) low potency of FR139317 and (3) non-competitive antagonism by BQ-123.

Heterogeneity of endothelin-sarafotoxin receptors mediating contraction of pig coronary artery.

The contractile effects of endothelin-1, endothelin-3, sarafotoxin 6b and sarafotoxin 6c were studied in endothelium-denuded rings of pig coronary artery. Endothelin-1, sarafotoxin 6b and sarafotoxin 6c produced monophasic concentration-response curves (mean EC50 values 6.7, 14.8 and 1.6 nM), whereas the concentration-response curve to endothelin-3 was biphasic (mean EC50 values 9.6 nM and 0.32 microM). The maximal effect of sarafotoxin 6c was about one third of that reached by the other peptides. The higher sensitivity component of the curve to endothelin-3 was abolished in the presence of sarafotoxin 6c (0.3 microM), while the EC50 value for the other component remained unchanged. Sarafotoxin 6c (0.3 microM) failed to alter the EC50 values of endothelin-1 and sarafotoxin 6b. These data strongly suggest the presence of at least two endothelin-sarafotoxin receptors mediating contraction of pig coronary artery, one with the profile of the endothelin ETA receptor subtype, the other recognizing sarafotoxin 6c and endothelin-3, but not endothelin-1 and sarafotoxin 6b, being thus different from the ETB receptor subtype.