Potential protein markers in children with Autistic Spectrum Disorder (ASD) revealed by salivary proteomics.

The lack of specific pharmacological therapy for Autistic Spectrum Disorder (ASD) and its clinical heterogeneity demand efforts directed toward the identification of biomarkers to aid in diagnosis. Proteomics offers a new perspective for studying the altered proteins associated with autism spectrum disorders (ASD) and we have saliva as an easy-to-collect biological fluid with important biomolecules for investigating biomarkers in various diseases. In this sense, saliva could be used to identify potential biomarkers of ASD. In the current work, saliva samples were collected from children with different degrees of ASD and healthy children and proteomics approaches were applied to generate data on differentially expressed proteins between groups which will serve as a basis for future validation studies as protein markers. Data are available via ProteomeXchange with identifier PXD030065. As results, 132 proteins were present in 80% of the saliva pools of all analyzed groups. Twenty-five proteins were identified as overexpressed in the group of severe and mild/moderate ASD carriers, among which, eight were identified as potential biomarkers for ASD.

Anethole Supplementation During Oocyte Maturation Improves In Vitro Production of Bovine Embryos.

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher (P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased (P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

Anethole Supplementation During Oocyte Maturation Improves In Vitro Production of Bovine Embryos.

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 µg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 µg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 µg/mL had higher ( P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased ( P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 µg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

2,2'-Azobis (2-Amidinopropane) Dihydrochloride Is a Useful Tool to Impair Lung Function in Rats.

Recently, several studies have reported that respiratory disease may be associated with an increased production of free radicals. In this context, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is a free radical-generating compound widely used to mimic the oxidative stress state. We aimed to investigate whether AAPH can generate lung functional, inflammatory, histological and biochemical impairments in the lung. Wistar rats were divided into five groups and instilled with saline solution (714 µL/kg, CTRL group) or different amounts of AAPH (25, 50, 100, and 200 mg/kg, 714 µL/kg, AAPH groups). Seventy-two hours later the animals were anesthetized, paralyzed, intubated and static elastance (Est), viscoelastic component of elastance (ΔE), resistive (ΔP1) and viscoelastic (ΔP2) pressures were measured. Oxidative damage, inflammatory markers and lung morphometry were analyzed. ΔP1 and Est were significantly higher in AAPH100 and AAPH200 than in the other groups. The bronchoconstriction indexes were larger in AAPH groups than in CTRL. The area occupied by collagen and elastic fibers, polymorpho- and mononuclear cells, malondialdehyde and carbonyl groups levels were significantly higher in AAPH200 than in CTRL. In comparison to CTRL, AAPH200 showed significant decrease and increase in the activities of superoxide dismutase and catalase, respectively. AAPH augmented the release of pro-inflammatory cytokines IL-1ß, IL-6 e TNF-α. Hence, exposure to AAPH caused significant inflammatory alterations and redox imbalance accompanied by altered lung mechanics and histology. Furthermore, we disclosed that exposure to AAPH may represent a useful in vivo tool to trigger lung lesions.

Eugenol dilates mesenteric arteries and reduces systemic BP by activating endothelial cell TRPV4 channels.

BACKGROUND AND PURPOSE: Eugenol, a vanilloid molecule found in some dietary plants, relaxes vasculature in part via an endothelium-dependent process; however, the mechanisms involved are unclear. Here, we investigated the endothelial cell-mediated mechanism by which eugenol modulates rat mesenteric artery contractility and systemic BP. EXPERIMENTAL APPROACH: The isometric tension of rat mesenteric arteries (size 200-300 µm) was measured using wire myography; non-selective cation currents (ICat ) were recorded in endothelial cells using patch clamp electrophysiology. Mean arterial pressure (MAP) and heart rate (HR) were determined in anaesthetized rats. KEY RESULTS: Eugenol relaxed endothelium-intact arteries in a concentration-dependent manner and this effect was attenuated by endothelium denudation. L-NAME, a NOS inhibitor, a combination of TRAM-34 and apamin, selective blockers of intermediate and small conductance Ca(2+) -activated K(+) channels, respectively, and HC-067047, a TRPV4 channel inhibitor, but not indomethacin, a COX inhibitor, reduced eugenol-induced relaxation in endothelium-intact arteries. Eugenol activated HC-067047-sensitive ICat in mesenteric artery endothelial cells. Short interfering RNA (siRNA)-mediated TRPV4 knockdown abolished eugenol-induced ICat activation. An i.v. injection of eugenol caused an immediate, transient reduction in both MAP and HR, which was followed by prolonged, sustained hypotension in anaesthetized rats. This sustained hypotension was blocked by HC-067047. CONCLUSIONS AND IMPLICATIONS: Eugenol activates TRPV4 channels in mesenteric artery endothelial cells, leading to vasorelaxation, and reduces systemic BP in vivo. Eugenol may be therapeutically useful as an antihypertensive agent and is a viable molecular candidate from which to develop second-generation TRPV4 channel activators that reduce BP.

Monoterpenoid terpinen-4-ol exhibits anticonvulsant activity in behavioural and electrophysiological studies.

Terpinen-4-ol (4TRP) is a monoterpenoid alcoholic component of essential oils obtained from several aromatic plants. We investigated the psychopharmacological and electrophysiological activities of 4TRP in male Swiss mice and Wistar rats. 4TRP was administered intraperitoneally (i.p.) at doses of 25 to 200 mg/kg and intracerebroventricularly (i.c.v.) at concentrations of 10, 20, and 40 ng/2 µL. For in vitro experiments, 4TRP concentrations were 0.1 mM and 1.0 mM. 4TRP (i.p.) inhibited pentylenetetrazol- (PTZ-) induced seizures, indicating anticonvulsant effects. Electroencephalographic recordings showed that 4TRP (i.c.v.) protected against PTZ-induced seizures, corroborating the behavioural results. To determine whether 4TRP exerts anticonvulsant effects via regulation of GABAergic neurotransmission, we measured convulsions induced by 3-mercapto-propionic acid (3-MP). The obtained results showed involvement of the GABAergic system in the anticonvulsant action exerted by 4TRP, but flumazenil, a selective antagonist of the benzodiazepine site of the GABAA receptor, did not reverse the anticonvulsant effect, demonstrating that 4TRP does not bind to the benzodiazepine-binding site. Furthermore, 4TRP decreased the sodium current through voltage-dependent sodium channels, and thus its anticonvulsant effect may be related to changes in neuronal excitability because of modulation of these channels.

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.

Relaxant effect of the essential oil of Croton nepetifolius on ovine cervix

We investigated the in vitro effect of the essential oil of Croton nepetifolius Baill., Euphorbiaceae (EOCN), on spontaneous or induced contractions of circular and longitudinal muscles from the ovine cervix during the luteal phase of the estrous cycle. The relaxant effect of EOCN was expressed as a percentage of the contraction recorded before the addition of the oil and calculated relative to the preparations exposed only to the vehicle. The IC50 (concentration of oil required to produce a 50% maximal reduction in muscle contraction) for relaxation of spontaneous contractions in circular and longitudinal muscles was significantly lower than the IC50 for blockade of K+-induced contraction (27.19 µg mL-1 versus 262.72 µg mL-1 and 40.92 µg mL-1 versus 222.47 µg mL-1, respectively). Interestingly, there was a high degree of selectivity in the action of EOCN on cervix layers concerning the inhibition of acetylcholine-induced contraction in circular (IC50 277.10 µg mL-1) and longitudinal (IC50 52.56 µg mL-1) muscles. In conclusion, EOCN is able to relax ovine cervix during the luteal phase. This work opens the perspective of applying EOCN in ovine embryo transfer.

Eugenol attenuates pulmonary damage induced by diesel exhaust particles.

Environmentally relevant doses of inhaled diesel particles elicit pulmonary inflammation and impair lung mechanics. Eugenol, a methoxyphenol component of clove oil, presents in vitro and in vivo anti-inflammatory and antioxidant properties. Our aim was to examine a possible protective role of eugenol against lung injuries induced by diesel particles. Male BALB/c mice were divided into four groups. Mice received saline (10 µl in; CTRL group) or 15 µg of diesel particles DEP (15 µg in; DIE and DEUG groups). After 1 h, mice received saline (10 µl; CTRL and DIE groups) or eugenol (164 mg/kg; EUG and DEUG group) by gavage. Twenty-four hours after gavage, pulmonary resistive (ΔP1), viscoelastic (ΔP2) and total (ΔPtot) pressures, static elastance (Est), and viscoelastic component of elastance (ΔE) were measured. We also determined the fraction areas of normal and collapsed alveoli, amounts of polymorpho- (PMN) and mononuclear cells in lung parenchyma, apoptosis, and oxidative stress. Est, ΔP2, ΔPtot, and ΔE were significantly higher in the DIE than in the other groups. DIE also showed significantly more PMN, airspace collapse, and apoptosis than the other groups. However, no beneficial effect on lipid peroxidation was observed in DEUG group. In conclusion, eugenol avoided changes in lung mechanics, pulmonary inflammation, and alveolar collapse elicited by diesel particles. It attenuated the activation signal of caspase-3 by DEP, but apoptosis evaluated by TUNEL was avoided. Finally, it could not avoid oxidative stress as indicated by malondialdehyde.

The essential oil of Eucalyptus tereticornis, and its constituents alpha- and beta-pinene, potentiate acetylcholine-induced contractions in isolated rat trachea.

The effects of the essential oil of Eucalyptus tereticornis (EOET), especially the effects of its constituents alpha- and beta-pinene, were studied on rat trachea in vitro. In tracheal rings, EOET, alpha- or beta-pinene potentiated the contractions induced by acetylcholine (ACh). Contractions induced by K(+) (60mM) were also potentiated by alpha- and beta-pinene, but were reduced by EOET. Our findings show that EOET has myorelaxant effects on rat airways, but potentiates ACh-induced contractions. Monoterpenes alpha- and beta-pinene are involved in its potentiating actions, but are not responsible for its myorelaxant effects. A putative inhibition of the acetylcholinesterase enzyme is involved.

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.

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.

Terpinen-4-ol: mechanisms of relaxation on rabbit duodenum.

The effect of terpinen-4-ol was studied on rabbit duodenum in-vitro. Terpinen-4-ol induced relaxation of the basal tonus (IC50 170.2 (95% confidence interval, 175-204) microM) with a maximal relaxant response of 180.4+/-3.9% (n=6) of the contraction induced by 60 mM [K(+)]. The maximal relaxation induced in control conditions was not affected (P>0.05) by pretreatment of the tissues with phentolamine (50 microM) or propranolol (10 microM), N(G) nitro-L-arginine methyl ester (L-NAME; 1 mM), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 100 microM), hexamethonium (1 mM), tetrodotoxin (1 microM), the mixture charybdotoxin-apamin (1 microM), glibenclamide (10 microM), 4-aminopyridine (10 microM) or tetraethyl-ammonium (100 microM). In addition, terpinen-4-ol completely relaxed tissues precontracted with 60 mM [K(+)] solutions (IC50 325.9 (245.1-433.1) microM) and also blocked (IC50 154.7 (117.7-191.7) microM) the phasic component of this contraction. At a concentration of 195 and 650 muM it reduced by 41.3+/-3.4% and 75.4+/-3.1%, respectively the maximal contractile response to Ca(2+) in depolarized duodenum. Terpinen-4-ol completely blocked the component of carbachol-induced contraction, which was resistant to nifedipine (100 microM) pretreatment or to a Ca(2+)-free solution. These data show that terpinen-4-ol relaxes intestinal smooth muscle and suggest that this effect is myogenic in nature and depends on calcium antagonism.

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.

Relaxant effects of the essential oil of Ocimum gratissimum on isolated ileum of the guinea pig.

The effects of the essential oil of Ocimum gratissimum L. (Labiatae) (EOOG) on guinea pig ileum were studied. EOOG (0.1-1000 microg/ml) reversibly and concentration-dependently relaxed the basal tone of the ileum and reversed the tonic contractions induced by 60 mM KCl and 10 microM acetylcholine, with IC(50) values of 23.8+/-5.2, 18.6+/-4.0 and 70.0+/-4.6 microg/ml, respectively. Our results show that EOOG exerts relaxant effects on intestinal smooth muscle, consistent with the popular use of the plant to treat gastrointestinal disorders.

Comparative inhibitory effects of niflumic acid and novel synthetic derivatives on the rat isolated stomach fundus.

Novel derivatives of 2-[3-(trifluoromethyl)-analino]nicotinic acid (niflumic acid) were synthesized. The compounds were compared for their inhibitory effects on 5-hydroxytryptamine (5-HT)- and KCI-induced contraction of the rat fundus. The aim was to assess structure-activity relationships regarding the selectivity and potency of these compounds. Niflumic acid (1-100 microM) concentration-dependently inhibited 5-HT-induced tonic contractions with an IC50 value (concentration reducing the control contractile response by 50%, calculated from semi-log graphs) of 0.24 x 10(4) M (n = 9). In contrast, it was significantly less potent at inhibiting KCl-induced responses (IC50 = 1.49 x 10(4) M, n = 9). The methyl ester (NFAme) and amido (NFAm) analogues showed no selectivity between 5-HT- and KCl-induced contractions with IC50 values of 1.64 x 10(-4) M (n = 8) and 1.87 x 10(-4) M (n = 9) for 5-HT responses, and 2.61 x 10(-4) M (n = 8) and 2.55 x 10(-4) M (n = 7) for KCl-induced responses, respectively. Our results suggest that alteration of the carboxylic acid moiety of niflumic acid reduces the selectivity and potency of its inhibitory action on 5-HT-induced contractile responses of the rat fundus, possibly via a reduced interaction with calcium-activated chloride channels.