Pedro Jorge Caldas Magalhães

Vasorelaxant effects of the monoterpenic phenol isomers, carvacrol and thymol, on rat isolated aorta

Various essential oils are rich in carvacrol, a monoterpenic phenol isomeric with thymol. This study was undertaken to assess the vasorelaxant effects of thymol and carvacrol in rat isolated aorta and the putative mechanisms underlying these effects. Thymol and carvacrol produced a concentration‐dependent relaxation on the aortic ring preparations pre‐contracted using KCl (IC50 value of 64.40 ± 4.41 and 78.80 ± 11.91 μm, respectively) or using phenylephrine (PHE, 0.1 μm) (IC50 value of 106.40 ± 11.37 and 145.40 ± 6.07 μm, respectively) and inhibited the concentration‐response curves of aortic rings to PHE or KCl. In Ca2+‐free medium with ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (2 mm), thymol and carvacrol both at 1000 μm completely abolished the phasic component of PHE‐induced endothelium‐containing ring contractions. At 400 μm, thymol and carvacrol significantly reduced the CaCl2‐induced contractions in Ca2+‐free medium. Furthermore, both thymol and carvacrol (300 and 1000 μm) significantly reduced the contraction evoked by phorbol dibutyrate (1 μm), an activator of protein kinase C. Magnitude of this inhibitory effect was enhanced in the presence of the Ca2+ pump inhibitor, thapsigargin (1 μm). At 1000 μm, neither thymol nor carvacrol altered the resting potential of vascular smooth muscle cells. In conclusion, thymol and carvacrol induced an endothelium‐independent relaxation in rat isolated aorta, an effect that seems mediated through some mechanisms probably involving a transduction pathway between Ca2+ release from sarcoplasmic reticulum and/or regulation of the Ca2+ sensitivity of the contractile system. Moreover, it’s conceivable that thymol and carvacrol, at low concentrations, block the Ca2+ influx through the membrane.

Intestinal myorelaxant and antispasmodic effects of the essential oil of Croton nepetaefolius and its constituents cineole, methyl-eugenol and terpineol

The effects of the essential oil of Croton nepetaefolius (EOCN), a medicinal plant from the north‐east of Brazil, and its constituents cineole, methyl‐eugenol and terpineol, were studied on intestinal motility in vivo and on in vitro mechanical activity of intestinal smooth muscle. In mice, EOCN (10–100 mg/kg body weight, intragastrically) increased the intestinal transit of charcoal marker delivered to the stomach. This was also observed in animals pretreated with castor oil. In segments of guinea‐pig ileum and cardial, pyloral and ileo‐caecal sphincters, EOCN preferentially decreased basal tonus compared with the amplitude of spontaneous contractions with EC50 values in the range 0.9 – 16 and 8 – 150 μg/mL respectively. In ileum, EOCN, cineole, methyl‐eugenol and terpineol decreased tonus with EC50 values of 16, 322, 9 and 71 μg/mL, respectively, and blocked 60 mM [K+]‐induced contraction with IC50 values of 18, 419, 12 and 95 μg/mL.

Linalool blocks excitability in peripheral nerves and voltage-dependent Na+ current in dissociated dorsal root ganglia neurons

Linalool is a terpene that occurs as a major constituent of essential oils of many plants of widespread distribution. It possesses several biological and pharmacological activities, including depressant effects on the central nervous system and olfactory receptors. The present study investigated whether linalool affects the excitability of peripheral components of the somatic sensory system. We used sciatic nerve and preparations of intact and dissociated neurons of dorsal root ganglion for extracellular, intracellular and patch-clamp recordings. Linalool concentration-dependently (0.3-2.0mM) and reversibly blocked the excitability of the sciatic nerve. It inhibited peak-to-peak amplitude of the compound action potential (IC(50) was 0.78+/-0.04 mM). At 0.8mM, it reversibly increased rheobase and chronaxy (from 3.2+/-0.1 V and 52.4+/-4.1 micros to 4.2+/-0.3 V and 71.2+/-5.5 micros (n=5), respectively) and inhibited with greater pharmacological potency the amplitude of the compound action potential components corresponding to axons with slower velocity of conduction. In a similar concentration range (0.1-6mM), linalool concentration-dependently and reversibly blocked the generation of action potentials of intact dorsal root ganglion neurons without alteration of resting membrane potential and input resistance, and inhibited the voltage-gated Na(+) current of dissociated dorsal root ganglion neurons. In conclusion, we demonstrated that linalool acts on the somatic sensory system with local anesthetic properties, since it blocked the action potential by acting on voltage-dependent Na(+) channels. This finding is important in showing the potential usefulness of linalool as a pharmacotherapeutic agent.

EFFECTS OF PIPERITENONE OXIDE ON THE INTESTINAL SMOOTH MUSCLE OF THE GUINEA PIG

We investigated the effects of piperitenone oxide (PO), a major constituent of the essential oil of Mentha x villosa, on the guinea pig ileum. PO (30 to 740 micrograms/ml) relaxed basal tonus without significantly altering the resting membrane potential. In addition, PO relaxed preparations precontracted with either 60 mM K+ or 5 mM tetraethylammonium in a concentration-dependent manner. At concentrations from 0.1 to 10 micrograms/ml PO potentiated acetylcholine-induced contractions, while higher concentrations (> 30 micrograms/ml) blocked this response. These higher PO concentrations also inhibited contractions induced by 60 mM K+. PO also blocked the components of acetylcholine contraction which are not sensitive to nifedipine or to solutions with nominal zero Ca2+ and EGTA. These results show that PO is a relaxant of intestinal smooth muscle and suggest that this activity may be mediated at least in part by an intracellular effect.

Inhaled 1,8-Cineole Reduces Inflammatory Parameters in Airways of Ovalbumin-Challenged Guinea Pigs

Eucalyptol, also known as 1,8-cineole, is a monoterpene traditionally used to treat respiratory disorders due to its secretolytic properties. In addition to its myorelaxant effects, it also has anti-inflammatory actions in vitro. In this study, we aimed to evaluate the efficacy of acute treatment with 1,8-cineole on reducing airway inflammatory parameters. Ovalbumin (OVA)-sensitized guinea pigs were submitted to antigenic challenge (OVA) with or without pre-treatment with a single dose of 1,8-cineole administered by inhalation. Airway inflammatory parameters were reduced or absent in 1,8-cineole-treated animals as compared with untreated guinea pigs. Acute treatment with 1,8-cineole impaired the development of airway hyperresponsiveness to carbachol in isolated tracheal rings. Levels of the pro-inflammatory cytokines TNFα and IL-1β was lower in bronchoalveolar lavage fluid (BALF) of 1,8-cineol-treated guinea pigs than in untreated animals. 1,8-Cineole impaired the OVA-induced increase of the myeloperoxidase activity in BALF. 1,8-Cineole also prevented the reduction of the mucociliary clearance induced by the antigen presentation. The present investigation provides evidence that inhaled 1,8-cineole prevents hyperresponsiveness and inhibits inflammation in airways of ovalbumin-challenged guinea pigs.

Effects of the essential oil of Croton zehntneri, and its constituent estragole on intestinal smooth muscle

The effects on isolated guinea‐pig ileum of the essential oil of Croton zehntneri (CZEO) and of its main constituent estragole (57% of CZEO by weight) were studied. CZEO and estragole (0.1–100 μg/mL) decreased the tonus in 56% and 61.5%, respectively, of the muscles. At concentrations above 10 μg/mL, they induced spontaneous rhythmic movements of small amplitude (less than 11% of the potassium contraction peak to peak). At concentrations from 1 to 100 μg/mL and with similar potencies, these agents blocked the contractions induced by acetylcholine, histamine and 50 mM K+ and caused relaxation of already established potassium contractures. Tested separately, CZEO, estragole and anethole (28% of CZEO by weight) blocked the contraction induced by Ca++ in the presence of 50 mM K+, but CZEO was more potent than estragole or anethole in blocking the Ca++‐induced contractions than those induced by K+. With large increases in the agonist concentration, the action of the oils on the contractions induced by Ca++ was reversible; however, their effect on contractions induced by histamine or ACh was not. The data show that the essential oil of Croton zehntneri has an effect on intestinal smooth muscle that is predominantly antispasmodic, and attributable in part to the effect of estragole, a major constituent.

Essential oil of croton nepetaefolius and its main constituent, 1,8-cineole, block excitability of rat sciatic nerve in vitro.

1 The effects of the essential oil of Croton nepetaefolius (EOCN) and its major constituent, 1,8‐cineole, on the compound action potential (CAP) of nerve were investigated. 2 Experiments were performed in sciatic nerves dissected from Wistar rats, mounted in a moist chamber and stimulated at a frequency of 0.2 Hz, with electric pulses of 100 µs duration at 20–40 V. Evoked CAP were displayed on an oscilloscope and recorded on a computer. The CAP control parameters were as follows: peak‐to‐peak amplitude 8.1 ± 0.6 mV (n = 15); conduction velocity 83.3 ± 4.2 m/s (n = 15); chronaxie 58.0 ± 6.8 msec (n = 6); and rheobase 2.8 ± 0.1 V (n = 6). 3 Lower concentrations of EOCN (100 and 300 µg/mL) and 1,8‐cineole (153 and 307 µg/mL; i.e. 1 and 2 mmol/L, respectively) had no significant effects on CAP control parameters throughout the entire recording period. However, at the end of 180 min exposure of the nerve to the drug, peak‐to‐peak amplitude was significantly (P < 0.05) reduced to 27.4 ± 6.7 and 1.7 ± 0.8% of control values by 500 and 1000 µg/mL EOCN, respectively (n = 6), and to 76.5 ± 4.4, 70.0 ± 3.9 and 14.8 ± 4.1% of control values by 614, 920 and 1227 µg/mL (i.e. 4, 6 and 8 mmol/L) 1,8‐cineole, respectively (n = 6). Regarding conduction velocity, at the end of the 180 min exposure period, this parameter was significantly reduced to 85.8 ± 7.3 and 48.7 ± 12.3% (n = 6) of control values by 500 and 1000 µg/mL EOCN, respectively, and to 86.4 ± 4.5 and 76.1 ± 5.2% (n = 6) by 920 and 1227 µg/mL 1,8‐cineole, respectively. Chronaxie and rheobase were significantly increased by the higher concentrations of both EOCN and 1,8‐cineole. 4 It is concluded that EOCN and its main constituent 1,8‐cineole block nerve excitability in a concentration‐dependent manner, an effect that was totally reversible with 1,8‐cineole but not with EOCN. This suggests that other constituents of EOCN, in addition to 1,8‐cineole, may contribute to the mediation of this effect of EOCN.

Pharmacological evidence of calcium-channel blockade by essential oil of Ocimum gratissimum and its main constituent, eugenol, in isolated aortic rings from DOCA-salt hypertensive rats

Intravenous (i.v.) treatment of conscious DOCA‐salt hypertensive rats with the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) induced a hypotensive effect that seems related to an active vascular relaxation rather than withdrawal of sympathetic tone. To corroborate this hypothesis, the present study examined the vascular effects of EOOG and its main constituent, eugenol (EUG) and the putative mechanisms underlying these effects. Additionally, the role of the vascular β2‐adrenergic mechanism in the mediation of EOOG‐induced hypotension has also been investigated. In conscious DOCA‐salt hypertensive rats, the EOOG‐induced hypotension was reversible and remained unchanged by i.v. pretreatment with propranolol (2 mg/kg). In isolated aorta preparations with intact endothelium from DOCA‐salt hypertensive rats, EOOG (1–1000 μg/mL) and EUG (0.006–6 mm) relaxed the phenylephrine‐induced contraction similarly with IC50 [geometric mean (95% confidence interval)] values of 226.9 (147.8–348.3) μg/mL and 1.2 (0.6–2.1) mm, respectively. Vasorelaxant effects of EOOG were significantly altered by removal of the vascular endothelium [IC50 = 417.2 (349.5–497.8) μg/mL]. In a calcium‐free medium, the CaCl2‐induced contractions were significantly reduced and even abolished by EOOG at 300 and 1000 μg/mL, respectively, whereas EOOG (1000 μg/mL) did not have any significant effect on caffeine‐induced contractions. Similar results were obtained with EUG (1.8 and 6 mm) on both CaCl2‐ and caffeine‐induced contractions, respectively. The data suggest that hypotensive responses to EOOG in DOCA‐salt hypertensive rats are due to an active vascular relaxation, which is partly dependent upon the integrity of the vascular endothelium and seems predominantly mediated through an inhibition of plasmalemmal Ca2+ influx rather than Ca2+‐induced Ca2+ release from the sarcoplasmic reticulum.

Cardiovascular effects of the essential oil of Aniba canelilla bark in normotensive rats.

Cardiovascular effects of intravenous (i.v.) treatment with the essential oil of the bark of Aniba canelilla (EOAC) were investigated in normotensive rats. In both pentobarbital-anesthetized and conscious rats, i.v. bolus injections of EOAC (1 to 20 mg/kg) elicited similar and dose-dependent hypotension and bradycardia. Pretreatment of anesthetized rats with bilateral vagotomy significantly reduced the bradycardia without affecting the hypotension. In conscious rats, pretreatment with hexamethonium (30 mg/kg, i.v.) significantly reduced the EOAC-induced bradycardia without affecting the hypotension. The opposite effect was observed after i.v. pretreatment with the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl esther (L-NAME, 20 mg/kg). However, both EOAC-induced hypotension and bradycardia were significantly reduced by pretreatment with methylatropine (1 mg/kg, i.v.). In rat endothelium-containing aorta preparations, EOAC (1-600 μg/mL) induced a concentration-dependent reduction of potassium (60 mM)-induced contraction [IC50 (geometric mean ± 95% confidence interval) = 64.5 (45.6-91.2) μg/mL)], an effect that was significantly reduced by the addition of atropine (10 μM) in the perfusion medium [IC50 = 109.5 (72.5-165.4) μg/mL)]. Furthermore, the vasorelaxant effects of the EOAC were also but significantly reduced [IC50 = 139.1 (105.2-183.9) μg/mL)] by removal of the vascular endothelium. Furthermore, the CaCl2-induced contractions in calcium-free medium were reduced and even fully abolished by EOAC (100 and 600 μg/mL), respectively. However, EOAC (600 μg/mL) was without significant effect on caffeine-induced contractions in calcium-free medium. These data show that i.v. treatment of rats with EOAC induces dose-dependent hypotension and bradycardia, which occurred independently. The bradycardia appears mainly dependent upon the presence of an operational and functional parasympathetic drive to the heart. However, the hypotension is due to an active vascular relaxation rather than withdrawal of sympathetic tone. This relaxation seems partly mediated by an endothelial L-arginine/nitric oxide pathway through peripheral muscarinic receptor activation (endothelium-dependent relaxation) and predominantly through an inhibition of calcium inward current (endothelium-independent relaxation).

Antispasmodic effects of essential oil of Pterodon polygalaeflorus and its main constituent β-caryophyllene on rat isolated ileum.

This study investigates the effects of essential oil of Pterodon polygalaeflorus (EOPP) and β‐caryophyllene (β‐CAR). EOPP and β‐CAR relaxed the basal tone of ileum smooth muscle in a concentration‐dependent manner (IC50s = 394.35 ± 62.12 and 68.65 ± 9.51 μg/mL respectively), an effect that was unaltered by hexamethonium, L‐nitroarginine methyl ester or indomethacin. Both EOPP and β‐CAR evoked a concentration‐dependent relaxation of ileum pre‐contracted with KCl with an IC50 value of 107.78 ± 10.47 and 17.35 ± 0.75 μg/mL, respectively. EOPP and β‐CAR inhibited the contractions induced by acetylcholine (ACh) and by KCl. In ileal preparations, the CaCl2‐induced contractions were reduced by EOPP (300 μg/mL) and β‐CAR (100 μg/mL). Furthermore, CaCl2‐induced contractions were also reduced by EOPP (300 μg/mL) and β‐CAR (100 μg/mL) in ileal preparations pretreated with ACh under Ca2+‐free condition and in the presence of verapamil. EOPP (100 and 300 μg/mL) and β‐CAR (30 and 100 μg/mL) reduced the ACh‐induced contractions of isolated rat ileum under Ca2+‐free conditions. In the presence of high KCl and Ca2+‐free conditions, EOPP (300 μg/mL) and β‐CAR (100 μg/mL) reduced the contractions induced by barium. A similar effect was also observed with verapamil. It is concluded that (i) β‐CAR is an important constituent involved in the myorelaxant and antispasmodic effects induced by EOPP; (ii) the inhibitory effect on intestinal contractility is myogenic and seems mainly mediated through an intracellular mechanism. However, the ability of EOPP and β‐CAR to decrease Ca2+ influx through cytoplasmic membrane could not be discounted.