The monoterpene 1,8-cineole prevents cerebral edema in a murine model of severe malaria.

1,8-Cineole is a naturally occurring compound found in essential oils of different plants and has well-known anti-inflammatory and antimicrobial activities. In the present work, we aimed to investigate its potential antimalarial effect, using the following experimental models: (1) the erythrocytic cycle of Plasmodium falciparum; (2) an adhesion assay using brain microvascular endothelial cells; and (3) an experimental cerebral malaria animal model induced by Plasmodium berghei ANKA infection in susceptible mice. Using the erythrocytic cycle of Plasmodium falciparum, we characterized the schizonticidal effect of 1,8-cineole. This compound decreased parasitemia in a dose-dependent manner with a half maximal inhibitory concentration of 1045.53 ± 63.30 µM. The inhibitory effect of 972 µM 1,8-cineole was irreversible and independent of parasitemia. Moreover, 1,8-cineole reduced the progression of intracellular development of the parasite over 2 cycles, inducing important morphological changes. Ultrastructure analysis revealed a massive loss of integrity of endomembranes and hemozoin crystals in infected erythrocytes treated with 1,8-cineole. The monoterpene reduced the adhesion index of infected erythrocytes to brain microvascular endothelial cells by 60%. Using the experimental cerebral malaria model, treatment of infected mice for 6 consecutive days with 100 mg/kg/day 1,8-cineole reduced cerebral edema with a 50% reduction in parasitemia. Our data suggest a potential antimalarial effect of 1,8-cineole with an impact on the parasite erythrocytic cycle and severe disease.

Eugenol disrupts Plasmodium falciparum intracellular development during the erythrocytic cycle and protects against cerebral malaria.

BACKGROUND: Malaria is a parasitic disease that compromises the human host. Currently, control of the Plasmodium falciparum burden is centered on artemisinin-based combination therapies. However, decreased sensitivity to artemisinin and derivatives has been reported, therefore it is important to identify new therapeutic strategies. METHOD: We used human erythrocytes infected with P. falciparum and experimental cerebral malaria (ECM) animal model to assess the potential antimalarial effect of eugenol, a component of clove bud essential oil. RESULTS: Plasmodium falciparum cultures treated with increasing concentrations of eugenol reduced parasitemia in a dose-dependent manner, with IC50 of 532.42 ± 29.55 µM. This effect seems to be irreversible and maintained even in the presence of high parasitemia. The prominent effect of eugenol was detected in the evolution from schizont to ring forms, inducing important morphological changes, indicating a disruption in the development of the erythrocytic cycle. Aberrant structural modification was observed by electron microscopy, showing the separation of the two nuclear membrane leaflets as well as other subcellular membranes, such as from the digestive vacuole. Importantly, in vivo studies using ECM revealed a reduction in blood parasitemia and cerebral edema when mice were treated for 6 consecutive days upon infection. CONCLUSIONS: These data suggest a potential effect of eugenol against Plasmodium sp. with an impact on cerebral malaria. GENERAL SIGNIFICANCE: Our results provide a rational basis for the use of eugenol in therapeutic strategies to the treatment of malaria.

Volatile oil of Croton zehntneri per oral sub-acute treatment offers small toxicity: perspective of therapeutic use

ABSTRACT Croton zehntneri Pax & K. Hoffm., Euphorbiaceae, or "canela-de-cunhé" is used in the Northeast Brazil to treat several diseases. Leaves and aerial parts of C. zehntneri are rich in volatile oil of high potential therapeutic. This study aimed to investigate volatile oil systemic toxicity after per oral treatment in rats. Volatile oil characterization (gas chromatography and mass spectrometry) showed 85.7% anethole and 4.8% estragole. Male Wistar rats (116-149 g) were treated with volatile oil (250 mg/kg p.o.) during ten weeks and evaluated for the following parameters: survival; food and water intake; body mass; absolute/relative organs weight; hemogram; plasma biochemical dosage; organs morphology. Volatile oil did not alter animal water and food consumption or the relative/absolute weight of most organs, but animals gained less weight. Volatile oil did not alter function biomarkers of pancreas, kidney, heart or liver, but increased plasma gamma-glutamyltranspeptidase (liver biomarker) and decreased uric acid (kidney biomarker). Although volatile oil had caused discrete morphological alterations in some organs, it did not induce architectural changes in these organs. In conclusion, the sub-acute per oral treatment with volatile oil no longer than ten weeks in rats offers small toxicity at doses below 250 mg/kg.

Essential oil of Croton zehntneri and its major constituent anethole display gastroprotective effect by increasing the surface mucous layer.

Croton zehntneri, a plant native to northeastern Brazil, is widely used in folk medicine to treat gastrointestinal problems and has rich essential oil content. The effects of the essential oil of Croton zehntneri (EOCZ) and its main constituent anethole on several models of gastric lesions were studied in mice and rats. Oral treatment with EOCZ and anethole, both at doses of 30-300 mg/kg, caused similar and dose-dependent gastroprotection against ethanol- and indomethacin-induced gastric damage, but did not change cold-restraint stress-induced ulcers in rats. Furthermore, EOCZ and anethole (both at 30 and 300 mg/kg) similarly and significantly increased the mucus production by the gastric mucosa, measured by Alcian blue binding, in ethanol-induced ulcer model. However, at the same doses, neither EOCZ nor anethole promoted significant alteration in gastric production of non-protein sulfhydryl groups. In pylorus-ligated model, neither EOCZ nor anethole (both at 30 and 300 mg/kg) had a significant effect on the volume of gastric juice, pH, or total acidity. The results of this study show for the first time that EOCZ possesses a gastroprotective potential, an effect mostly attributed to the action of anethole. This activity is related predominantly to the ability of EOCZ and anethole to enhance the production of gastric wall mucus, an important gastroprotective factor. Furthermore, they suggest that EOCZ has potential therapeutic application for the treatment of gastric ulcers.

Comparative study of the anti-edematogenic effects of anethole and estragole.

BACKGROUND: Anethole and estragole are monoterpene position isomers and constituents of essential oils from aromatic plants and were used in this study with the aim of analyzing their anti-inflammatory activity. METHODS: The anti-edematogenic effects of anethole and estragole were evaluated through plethysmometry in Swiss mice. RESULTS: Anethole inhibited carrageenan-induced edema at doses of 3, 10 and 30 mg/kg from 60 to 240 min after induction. However, the inhibitory effects of estragole were observed only from 60 to 120 min at the two highest doses. Anethole and estragole similarly inhibited edema elicited by substance P, bradykinin, histamine and TNF-α but were different in the inhibition of serotonin-elicited edema. In addition, only estragole inhibited sodium nitroprusside-induced edema. CONCLUSIONS: Anethole and estragole showed different profiles in the anti-inflammatory response to substance P, bradykinin, histamine, serotonin and TNF-α NO is involved only in the inhibition mechanism of estragole.

Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons.

Eugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na+ channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications, eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability.

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

This study investigates the effects of essential oil of Pterodon polygalaeflorus (EOPP) and ß-caryophyllene (ß-CAR). EOPP and ß-CAR relaxed the basal tone of ileum smooth muscle in a concentration-dependent manner (IC(50) s = 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 ß-CAR evoked a concentration-dependent relaxation of ileum pre-contracted with KCl with an IC(50) value of 107.78 ± 10.47 and 17.35 ± 0.75 µg/mL, respectively. EOPP and ß-CAR inhibited the contractions induced by acetylcholine (ACh) and by KCl. In ileal preparations, the CaCl(2) -induced contractions were reduced by EOPP (300 µg/mL) and ß-CAR (100 µg/mL). Furthermore, CaCl(2) -induced contractions were also reduced by EOPP (300 µg/mL) and ß-CAR (100 µg/mL) in ileal preparations pretreated with ACh under Ca(2+) -free condition and in the presence of verapamil. EOPP (100 and 300 µg/mL) and ß-CAR (30 and 100 µg/mL) reduced the ACh-induced contractions of isolated rat ileum under Ca(2+) -free conditions. In the presence of high KCl and Ca(2+) -free conditions, EOPP (300 µg/mL) and ß-CAR (100 µg/mL) reduced the contractions induced by barium. A similar effect was also observed with verapamil. It is concluded that (i) ß-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 ß-CAR to decrease Ca(2+) influx through cytoplasmic membrane could not be discounted.

Endothelium-dependent vasorelaxant effects of the essential oil from aerial parts of Alpinia zerumbet and its main constituent 1,8-cineole in rats.

Vasorelaxant effects of essential oil of Alpinia zerumbet (EOAZ) and its main constituent, 1,8-cineole (CIN) were studied. In rat isolated aorta preparations with intact endothelium, EOAZ (0.01-3000 microg/ml) induced significant but incomplete relaxation of the phenylephrine-induced contraction, an effect that was abolished by removal of vascular endothelium. However, at the same concentrations (0.01-3000 microg/ml corresponding to 0.0000647-19.5 mM), CIN induced a complete vasorelaxant effects (IC(50)=663.2+/-63.8 microg/ml) that were significantly reduced in endothelium-denuded rings (IC(50)=1620.6+/-35.7 microg/ml). Neither EOAZ nor CIN affected the basal tonus of isolated aorta. Vasorelaxant effects of both EOAZ and CIN remained unaffected by the addition of tetraethylamonium chloride (500 microM) or indomethacin (10 microM) into the bath, but were significantly reduced by N(G)-nitro-L-arginine methyl ester (100 microM). It is concluded that EOAZ induces a potent vasorelaxant effect that could not be fully attributed to the actions of the main constituent CIN, and appears totally dependent on the integrity of a functional vascular endothelium. The data is novel and corroborate the popular use of A. zerumbet for the treatment of hypertension.

Effects of 1,8-cineole on electrophysiological parameters of neurons of the rat superior cervical ganglion.

1. 1,8-Cineole is a non-toxic small terpenoid oxide believed to have medicinal properties in folk medicine. It has been shown to have various pharmacological effects, including blockade of the compound action potential (AP). In the present study, using intracellular recording techniques, we investigated the effects of 1,8-cineole on the electrophysiological parameters of neurons of the superior cervical ganglion (SCG) in rats. 2. 1,8-Cineole (0.1-6 mmol/L) showed reversible and concentration-dependent effects on various electrophysiological parameters. At 3 and 6 mmol/L, but not at 0.1 and 1 mmol/L, 1,8-cineole significantly diminished the input resistance (R(i)) and altered the resting potential (E(m)) to more positive values. At 6 mmol/L, 1,8-cineole completely blocked all APs within 2.7 +/- 0.6 min (n = 12). In neurons exposed to 3 and 1 mmol/L 1,8-cineole, the effects regarding excitability varied from complete AP blockade to minor inhibition of AP parameters. The depolarization of E(m) and the decrease in R(i) induced by 6 mmol/L 1,8-cineole were unaltered by 200 micromol/L niflumic acid, a well known blocker of Ca(2+)-activated Cl(-) currents. 3. Significant correlations (Pearson correlation test) were found between changes in E(m) and decreases in AP amplitude (r = -0.893; P < 0.00282) and maximum ascendant inclination (r = -0.799; P < 0.0173), but not for maximum descendant inclination (r = 0.598; P < 0.117). Application of current to restore the transmembrane potential equal to control E(m) values in the presence of 6 mmol/L 1,8-cineole resulted in the partial recovery of AP. 4. The present study shows that 1,8-cineole effectively blocks the excitability of SCG neurons, probably through various mechanisms, one of which acts indirectly via depolarization of the neuronal cytoplasmatic membrane.

Essential oil of Pterodon polygalaeflorus inhibits electromechanical coupling on rat isolated trachea.

The present work studied the effects of the essential oil of Pterodon polygalaeflorus (EOPP), a plant used to treat bronchitis and amigdalytis, on rat airway smooth muscle in vitro. In Ca(2+)-containing medium, EOPP (100-1300 microg/ml) inhibited preferentially high KCl- than 5-HT-induced muscle contractions in a concentration-dependent fashion, but did affect neither basal muscle tension nor ACh-induced contractions. In preparations maintained in either 60 mM K(+) or 10 microM ACh in Ca(2+)-free medium, EOPP (100, 600 and 1300 microg/ml) inhibited maximum contractile response induced by cumulative Ca(2+) addition (0.1-20 mM). Verapamil (10, 30 and 100 microg/ml), a Ca(2+) channel blocker, also inhibited Ca(2+)-induced concentration-effect curve in presence of ACh in Ca(2+)-free medium, whilst it was ineffective to decrease cholinergic contractions in Ca(2+)-containing medium. In presence of 150 mM K(+) in Ca(2+)-containing medium, EOPP (1300 microg/ml) did not reversed ACh-induced contractions. In contrast, under similar conditions, EOPP almost fully relaxed cholinergic contractions of tracheal smooth muscle in Ba(2+)-containing medium. In medium containing 10 mM tetraethylammonium and 2 mM Ba(2+) instead of Ca(2+), both EOPP (1300 microg/ml) and verapamil (approximately 5 microg/ml) significantly decreased ACh-induced contractions. Thus, in rat isolated trachea, EOPP induces inhibitor effects on contractions preferentially triggered by an electromechanical coupling mode.

Inhibitory actions of eugenol on rat isolated ileum.

The effects of eugenol (1-2000 microM) on rat isolated ileum were studied. Eugenol relaxed the basal tonus (IC50 83 microM) and the ileum precontracted with 60 mM KCl (IC50 162 microM), an action unaltered by 0.5 microM tetrodotoxin, 0.2 mM N(G)-nitro-L-arginine methyl ester, 0.5 mM hexamethonium, and 1 microM indomethacin. Eugenol did not alter the resting transmembrane potential (Em) of the longitudinal muscle layer under normal conditions (5.0 mM K+) or in depolarised tissues. Eugenol reversibly inhibited contractions induced by submaximal concentrations of acetylcholine (ACh) and K+ (40 mM) with IC50 values of approximately 228 and 237 microM, respectively. Eugenol blocked the component of ACh-induced contraction obtained in Ca(2+)-free solution (0.2 mM EGTA) or in the presence of nifedipine (1 microM). Our results suggest that eugenol induces relaxation of rat ileum by a direct action on smooth muscle via a mechanism largely independent of alterations of Em and extracellular Ca2+ influx.