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The pharmacological effects of Bothrops neuwiedi pauloensis venom on mouse phrenic nerve-diaphragm (PND) preparations were studied. Venom (20 mug/ml) irreversibly inhibited indirectly evoked twitches in PND preparations (60 ± 10% inhibition, mean ± SEM; p

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 µg/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 µg/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 µM acetylcholine alone and cumulative concentrations of 1 µM to 10 mM were unaffected. At venom concentrations higher than 50 µg/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24ºC, the venom (50 µg/ml) produced only partial neuromuscular blockade (30.7 ± 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Cuphea carthagenensis (Jacq.) J. F. Macbr. is a popular plant in Brazilian folk medicine owing to its hypotensive and central nervous system depressant effects. This study aimed to validate the hypotensive effect of the plant's aqueous extract (AE) in rats and examine the vascular actions of three hydrolyzable tannins, oenothein B, woodfordin C, and eucalbanin B, isolated from AE. Systolic blood pressure in unanesthetized rats was determined using the non-invasive tail-cuff method. Oral treatment of normotensive rats with 0.5 and 1.0 g/kg/day AE induced a dose-related hypotensive effect after 1 week. In rat aortic rings pre-contracted with noradrenaline, all ellagitannins (20 - 180 µM) induced a concentration-related vasorelaxation. This effect was blocked by either removing the endothelium or pre-incubating with N G -nitro-l-arginine methyl ester (10 µM), an inhibitor of nitric oxide (NO) synthase. In KCl-depolarized rat portal vein preparations, the investigated compounds did not affect significantly the maximal contractile responses and pD2 values of the concentration-response curves to CaCl 2 . Our results demonstrated the hypotensive effect of C. carthagenensis AE in unanesthetized rats. All isolated ellagitannins induced vasorelaxation in vitro via activating NO synthesis/NO release from endothelial cells, without altering the Ca 2+ influx in vascular smooth muscle preparations. Considering the low oral bioavailability of ellagitannins, the determined in vitro actions of these compounds are unlikely to account for the hypotensive effect of AE in vivo . It remains to be determined the role of the bioactive ellagitannin-derived metabolites in the hypotensive effect observed after oral treatment of unanesthetized rats with the plant extract., Competing Interests: The authors declare that they have no conflicts of interest., (Thieme. All rights reserved.)

Alcohol Use Disorder (AUD) presents a significant and challenging public health concern, marked by a dearth of effective pharmacological treatments. Understanding the neurobiological underpinnings of AUD is of paramount importance for the development of efficacious interventions. The process of addiction entails the acquisition of associative behaviors, prominently engaging the dorsal region of the hippocampus for encoding these associative memories. Nicotinic receptor systems have been implicated in mediating the rewarding effects of ethanol, as well as memory and learning processes. In our current investigation, we delved into the role of α4β2 nicotinic acetylcholine receptors (nAChRs) within the dorsal hippocampus in the context of ethanol-induced conditioned place preference (CPP), a robust model for scrutinizing the rewarding properties and drug-associated behaviors. To establish CPP, ethanol (2 g/kg) was administered intraperitoneally during a 8-day conditioning phase. Fos immunohistochemistry was employed to assess the involvement of discrete subregions within the dorsal hippocampus in ethanol-induced CPP. Additionally, we probed the influence of α4β2 nAChRs on CPP via microinjections of a selective nAChR antagonist, dihydro-β-erythroidine (DHBE, at dosages of 6, 12, and 18 µg/0.5 µL per hemisphere) within the hippocampus. Our results unveiled that ethanol-induced CPP was associated with an increase Fos -positive cells in various subregions of the dorsal hippocampus, including CA1, CA2, CA3, and the dentate gyrus. Intrahippocampal administration of DHBE (at doses of 6 and 18 µg/0.50 µL per hemisphere) effectively blocked ethanol-induced CPP, while leaving locomotor activity unaffected. These findings underscore the critical involvement of the dorsal hippocampus and α4β2 nAChRs in the acquisition of ethanol-associated learning and reward., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Proteases play a pivotal role in many signaling pathways; inhibitors of well-established proteases have shown a substantial therapeutic success. This study aimed to examine the in vivo effects of 3 protease inhibitors isolated from Bauhinia species: i) Bauhinia mollis elastase inhibitor, which blocks human neutrophil elastase (Ki app  2.8 nM) and cathepsin G (Ki app  1.0 nM) activities; ii) Bauhinia mollis trypsin inhibitor, a trypsin inhibitor (Ki app  5.0 nM); and iii) Bauhinia bauhinioides cruzipain inhibitor, which inhibits elastase (Ki app  2.6 nM), cathepsin G (Ki app  160.0 nM), and the cysteine proteases cathepsin L (Ki app  0.2 nM). Bauhinia bauhinioides cruzipain inhibitor, Bauhinia mollis elastase inhibitor, and Bauhinia mollis trypsin inhibitor were isolated using acetone and ammonium sulfate fractionations, DEAE-Sephadex, trypsin-Sepharose, and Resource-Q chromatographies. Mice and rats were treated intraperitoneally with 1 dose of inhibitor; gastric mucosal lesions were induced by cold-restraint stress. Oral pretreatment of mice with Bauhinia mollis elastase inhibitor or Bauhinia mollis trypsin inhibitor (1 - 10 mg/kg) did not show anti-ulcer effect, while Bauhinia bauhinioides cruzipain inhibitor (0.1 - 1.0 mg/kg) produced a similar reduction of the index of mucosal damage at all effective doses (30 to 33% < control). In rats at doses lower than those used in mice, Bauhinia mollis elastase inhibitor and Bauhinia bauhinioides cruzipain inhibitor reduced the index of mucosal damage by 66% and 54% of controls, respectively. The results indicate a protective effect against gastric mucosal lesions associated with elastase inhibition but not inhibition of trypsin activities. Moreover, the lack of Bauhinia mollis elastase inhibitor efficacy observed in mice may possibly be related to the reported structural differences of elastase in mice and rats., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Dystrophin deficiency caused by mutations of the related gene leads to muscle wasting in Duchenne muscular dystrophy (DMD). Some patients with DMD also present with intellectual disability and various degrees of neurological disorders, which have been related to a decreased number of postsynaptic gamma-aminobutyric acid type A receptors (GABA A Rs) in the hippocampus (HPC) and cerebellum (CBL). The aim of this study was to examine the relevance of dystrophin in the presynaptic GABAergic function in brain regions in which this protein is normally abundant. [ 3 H]-GABA release, induced by nicotinic receptor (nAChR) activation or K + depolarization, and [ 3 H]-GABA uptake were determined using synaptosomes extracted from the cortex (CTX), HPC, and CBL of littermate control and mdx mice. Superfusion of the synaptosomes with nicotine or high K + solutions led to a concentration-dependent and Ca 2+ -dependent [ 3 H]-GABA release in control and mdx synaptosomes. [ 3 H]-GABA release induced by 10 μM nicotine in mdx CBL synaptosomes was 47% less than that in control mice. K + -induced [ 3 H]-GABA release did not differ between control and mdx synaptosomes. α7-containing and β2-containing nAChRs were involved in nicotine-induced [ 3 H]-GABA release in control and mdx synaptosomes. Kinetic analysis of [ 3 H]-GABA uptake in mdx CBL synaptosomes showed a reduced (50%) half-maximal uptake time (t 1/2 ) and increased (44%) rate of [ 3 H]-GABA uptake (V max ) compared to controls. The apparent transporter affinity (K m ) for GABA was not altered. Our findings show that dystrophin deficiency in mdx mice is associated with significant changes in the release and uptake of GABA in the CBL. These presynaptic alterations may be related to the reported decrease in postsynaptic GABA A R in the same brain region. The results indicate possible dysfunction of GABAergic synapses associated with dystrophin deficiency in the CBL, which may contribute to the cognitive and neurobehavioral disorders in mdx mice and patients with DMD., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Tripeptidyl peptidase I (TPP-I), also named ceroid lipofuscinosis 2 protease (CLN2p), is a serine carboxyl lysosomal protease involved in neurodegenerative diseases, and has both tripeptidyl amino- and endo- peptidase activities under different pH conditions. We developed fluorescence resonance energy transfer (FRET) peptides using tryptophan (W) as the fluorophore to study TPP-I hydrolytic properties based on previous detailed substrate specificity study (Tian Y. et al., J. Biol. Chem. 2006, 281:6559-72). Tripeptidyl amino peptidase activity is enhanced by the presence of amino acids in the prime side and the peptide NH2-RWFFIQ-EDDnp is so far the best substrate described for TPP-I. The hydrolytic parameters of this peptide and its analogues indicated that the S4 subsite of TPP-I is occluded and there is an electrostatic interaction of the positively charged substrate N-terminus amino group and a negative locus in the region of the enzyme active site. KCl activated TPP-I in contrast to the inhibition by Ca(2+) and NaCl. Solvent kinetic isotope effects (SKIEs) show the importance of the free N-terminus amino group of the substrates, whose absence results in a more complex solvent-dependent enzyme: substrate interaction and catalytic process. Like pure TPP-I, rat spleen and kidney homogenates cleaved NH2-RWFFIQ-EDDnp only at F-F bond and is not inhibited by pepstatin, E-64, EDTA or PMSF. The selectivity of NH2-RWFFIQ-EDDnp to TPP-I was also demonstrated by the 400 times higher k(cat)/K(M) compared to generally used substrate, NH2-AAF-MCA and by its resistance to hydrolysis by cathepsin D that is present in high levels in kidneys., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Abnormalities in skeletal muscle structure and function are important contributors to exercise intolerance and functional decline in peripheral arterial disease. In this study, we tested the hypothesis that administration of N-acetylcysteine (NAC) would improve fatigue resistance and ameliorate the histopathological changes in skeletal muscle in a mouse model of peripheral arterial disease. We also anticipated that NAC treatment would lower the levels of biomarkers of oxidative damage in the ischemic muscle., Methods: Male Balb/c mice were subjected to bilateral ligation of the femoral artery and, after 2 weeks of recovery, received daily intraperitoneal injections of either NAC (150 mg/kg) or saline for 15 days. At the end of the treatment, the extensor digitorium longus (EDL) and soleus muscles were excised for assessment of contractile function in vitro and histological analysis. Free malondialdehyde and protein carbonyl levels were measured in the gastrocnemius muscle., Results: In the soleus muscle, force after 10 minutes of submaximal tetanic stimulation (60 Hz, 300 ms trains, 0.3 trains/s) was higher (P < .05) in NAC-treated animals (45% ± 3% of the initial value; n = 7) when compared with controls (30.3% ± 3%; n = 8). No differences were found in fatigue development between groups in the EDL muscle (ligated NAC, 35.7% ± 1.9%; ligated saline, 37.5% ± 1.1%). In addition, there was a tendency for lower levels of connective tissue deposition in the soleus of animals treated with NAC (n = 6) when compared with those that received only saline (n = 9) (ligated NAC, 16% ± 2% vs ligated saline, 24% ± 2%; P = .057). No differences were found in lipid peroxidation or protein carbonyl levels between ligated saline and ligated NAC groups., Conclusions: Taken together, these results indicate that treatment with NAC improves fatigue resistance in the soleus but not the EDL muscle in a model of peripheral arterial insufficiency., Clinical Relevance: Despite the increasing burden of peripheral arterial disease (PAD) and its detrimental consequences on the quality of life of the patients, few pharmacological therapies have shown to evoke meaningful effects on functional performance in these individuals. N-acetylcysteine is approved for clinical use, has minimal side effects and most important, has shown to consistently improve exercise performance in animals and humans. In this study, we showed, for the first time, that treatment with this drug at a dose amenable for clinical application evoked marked effects on fatigue resistance in the soleus muscle in a mouse model of PAD. These encouraging findings set the stage for translational studies to determine the acute and long-term impact of this drug on walking capacity in patients with PAD., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Baccharis trimera (Less.) D.C. (Asteraceae) is a medicinal species native to South America and used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. The aqueous extract (AE) of the aerial parts of this species presented two mainly constituents: the ent-clerodane diterpene (Fig. 1) and the neo-clerodane diterpene (Fig. 2). The objective of this work was to study their activities on the blockade of Ca(2+)-induced contractions in KCL-depolarized rat portal vein preparations, and on the influx and mobilization of cytosolic calcium in rat cardiomyocytes by fluorescence measurements. The results showed that both the neo- and the ent-clerodane diterpenes reduced the maximal contractions induced by CaCl2, in KCl depolarized rat portal vein preparations, without modifying the EC50. The data on the concentration of cytosolic calcium ([Ca(2+)]c) showed that, while the neo-clerodane diterpene stimulates the mobilization of [Ca(2+)]c in rat cardiomyocytes, this effect was not observed with the ent-clerodane diterpene. On the other hand, the influx of calcium was not altered by the neo-clerodane diterpene, but was reduced in the presence of the ent-clerodane diterpene, indicating that this compound induces a blockade of the voltage-dependent calcium channels., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Mild cognitive impairments have been described in one-third of patients with Duchenne muscle dystrophy (DMD). DMD is characterized by progressive and irreversible muscle degeneration caused by mutations in the dystrophin gene and lack of the protein expression. Previously, we have reported altered concentrations of α7- and β2-containing nicotinic acetylcholine receptors (nAChRs) in hippocampal membranes of dystrophic (mdx) mice. This suggests that alterations in the central cholinergic synapses are associated with dystrophin deficiency. In this study, we examined the release of acetylcholine (ACh) and the level of the vesicular ACh transporter (VAChT) using synaptosomes isolated from brain regions that normally have a high density of dystrophin (cortex, hippocampus and cerebellum), in control and mdx mice at 4 and 12months of age. ACh release evoked by nicotinic stimulation or K(+) depolarization was measured as the tritium outflow from superfused synaptosomes preloaded with [(3)H]-choline. The results showed that the evoked tritium release was Ca(2+)-dependent and mostly formed by [(3)H]-ACh. β2-containing nAChRs were involved in agonist-evoked [(3)H]-ACh release in control and mdx preparations. In hippocampal synaptosomes from 12-month-old mdx mice, nAChR-evoked [(3)H]-ACh release increased by 57% compared to age-matched controls. Moreover, there was a 98% increase in [(3)H]-ACh release compared to 4-month-old mdx mice. [(3)H]-ACh release evoked by K(+) depolarization was not altered, while the VAChT protein level was decreased (19%) compared to that of age-matched controls. In cortical and cerebellar preparations, there was no difference in nAChR-evoked [(3)H]-ACh release and VAChT levels between mdx and age-matched control groups. Our previous findings and the presynaptic alterations observed in the hippocampi of 12-month-old mdx mice indicate possible dysfunction of nicotinic cholinergic synapses associated with dystrophin deficiency. These changes may contribute to the cognitive and behavioral abnormalities described in dystrophic mice and patients with DMD., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Protein dystrophin is a component of the dystrophin-associated protein complex, which links the contractile machinery to the plasma membrane and to the extracellular matrix. Its absence leads to a condition known as Duchenne muscular dystrophy (DMD), a disease characterized by progressive skeletal muscle degeneration, motor disability, and early death. In mdx mice, the most common DMD animal model, loss of muscle cells is observed, but the overall disease alterations are less intense than in DMD patients. Alterations in gastrointestinal tissues from DMD patients and mdx mice are not yet completely understood. Thus, we investigated the possible relationships between morphological (light and electron microscopy) and contractile function (by recording the isometric contractile response) with alterations in Ca²⁺ handling in the ileum of mdx mice. We evidenced a 27% reduction in the ileal muscular layer thickness, a partial damage to the mucosal layer, and a partial damage to mitochondria of the intestinal myocytes. Functionally, the ileum from mdx presented an enhanced responsiveness during stretch, a mild impairment in both the electromechanical and pharmacomechanical signaling associated with altered calcium influx-induced contraction, with no alterations in the sarcoplasmic reticulum Ca²⁺ storage (maintenance of the caffeine and thapsigargin-induced contraction) compared with control animals. Thus, it is evidenced that the protein dystrophin plays an important role in the preservation of both the microstructure and ultrastructure of mice intestine, while exerting a minor but important role concerning the intestinal contractile responsiveness and calcium handling.

Lack of dystrophin in Duchenne muscle dystrophy (DMD) and in the mutant mdx mouse results in progressive muscle degeneration, structural changes at the neuromuscular junction, and destabilization of the nicotinic acetylcholine receptors (nAChRs). One-third of DMD patients also present non-progressive cognitive impairments. Considering the role of the cholinergic system in cognitive functions, the number of nAChR binding sites and the mRNA levels of α4, β2, and α7 subunits were determined in brain regions normally enriched in dystrophin (cortex, hippocampus and cerebellum) of mdx mice using specific ligands and reverse-transcription polymerase chain reaction assays, respectively. Membrane preparations of these brain regions were obtained from male control and mdx mice at 4 and 12 months of age. The number of [³H]-cytisine (α4β2) and [¹²⁵I]-α-bungarotoxin ([¹²⁵I]-αBGT, α7) binding sites in the cortex and cerebellum was not altered with age or among age-matched control and mdx mice. A significant reduction in [³H]-cytisine (48%) and [¹²⁵I]-αBGT (37%) binding sites was detected in the hippocampus of mdx mice at 12 months of age. When compared with the age-matched control groups, the mdx mice did not have significantly altered [³H]-cytisine binding in the hippocampus, but [¹²⁵I]-αBGT binding in the same brain region was 52% higher at 4 months and 20% lower at 12 months. mRNA transcripts for the nAChR α4, β2, and α7 subunits were not significantly altered in the same brain regions of all animal groups. These results suggest a potential alteration of the nicotinic cholinergic function in the hippocampus of dystrophin-deficient mice, which might contribute to the impairments in cognitive functions, such as learning and memory, that have been reported in the dystrophic murine model and DMD patients., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study investigates the gastroprotective effects of hecogenin, a steroid saponin isolated from Agave sisalana, on experimental models of gastric ulcer. Male Swiss mice were used in the models of ethanol- and indometacin-induced gastric ulcer. To clarify the hecogenin mechanism of action, the roles of nitric oxide (NO), sulfhydryls (GSH), K⁺(ATP) channels and prostaglandins were also investigated, and measurements of lipid peroxidation (TBARS assay) and nitrite levels in the stomach of hecogenin-treated and untreated animals were performed. Furthermore, the effects of hecogenin on myeloperoxidase (MPO) release from human neutrophils were assessed in vitro. Our results showed that hecogenin (3.1, 7.5, 15, 30, 60 and 90 mg/kg, p.o.) acutely administered, before ethanol or indomethacin, exhibited a potent gastroprotective effect. Although the pretreatments with L-NAME, an iNOS inhibitor, and capsazepine, a TRPV1 receptor agonist, were not able to reverse the hecogenin effect, this was reversed by glibenclamide, a K⁺(ATP) blocker, and indomethacin in the model of ethanol-induced gastric lesions. The hecogenin pretreatment normalized GSH levels and significantly reduced lipid peroxidation and nitrite levels in the stomach, as evaluated by the ethanol-induced gastric lesion model. The drug alone increased COX-2 expression and this effect was further enhanced in the presence of ethanol. It also decreased MPO release and significantly protected the gastric mucosa. In conclusion, we showed that hecogenin presents a significant gastroprotective effect that seems to be mediated by K⁺(ATP) channels opening and the COX-2/PG pathway. In addition, its antioxidant and anti-inflammatory properties may play a role in the gastroprotective drug effect., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Baccharis trimera (Less.) DC. (Asteraceae) is a species native to South America used in Brazilian folk medicine to treat gastrointestinal and liver diseases, kidney disorders and diabetes. Previous studies from this laboratory confirmed the antacid and antiulcer activities of the plant aqueous extract (AE) in rat and mouse models., Aim of the Study: To investigate the mechanisms involved in the antacid action of AE and isolated compounds from Baccharis trimera., Materials and Methods: AE was assayed in vivo in cold-restraint stress gastric ulcers and in pylorus-ligated mice. Nine fractions (F2-F10) previously isolated from AE were assayed in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands, and on gastric microsomal H(+), K(+)-ATPase preparations. Chlorogenic acids (F2, F3, F6, F7), flavonoids (F9), an ent-clerodane diterpene (F8) and a dilactonic neo-clerodane diterpene (F10) have been identified in these fractions., Results: Intraduodenal injection of AE (1.0 and 2.0 g/kg) in 4h pylorus-ligated mice decreased the volume (20 and 50%) and total acidity (34 and 50%) of acid secretion compared to control values. Administered orally at the same doses AE protected against gastric mucosal lesions induced in mice by restraint at 4°C. Exposure of isolated rabbit gastric glands to fractions F8 (10-100 μM) and F9 (10-300 μg/ml) decreased the basal [(14)C]-AP uptake by 50 and 60% of control (Ratio=6.2±1.1), whereas the remaining fractions were inactive. In the presence of the secretagogues F2 and F4 (30-300 μg/ml) decreased the [(14)C]-AP uptake induced by histamine (His) with a 100-fold lower potency than that of ranitidine. F5 and F6 reduced the [(14)C]-AP uptake stimulated by carbachol (CCh), but they were 10 to 20-fold less potent than atropine. F8 (diterpene 2) and F9 (flavonoids) decreased both the His- and CCh-induced [(14)C]-AP uptake, whereas F10 (diterpene 1) was inactive against the [(14)C]-AP uptake stimulated by secretagogues. Diterpene 2 was the most active of all tested compounds being 7-fold less potent than ranitidine and equipotent to atropine in reducing acid secretion in vitro. This compound also reduced the gastric H(+), K(+)-ATPase activity by 20% of control, while the remaining fractions were inactive on the proton pump in vitro., Conclusions: The results indicate that Baccharis trimera presents constituents that inhibit gastric acid secretion by acting mainly on the cholinergic regulatory pathway. The plant extract also contains compounds that exert moderate inhibition of the histaminergic regulatory pathway of acid secretion and the gastric proton pump. Altogether these active constituents appear to provide effective inhibition of acid secretion in vivo, which may explain the reputed antiulcer activity of the plant extract., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Ethnopharmacological Relevance: The leaves of Piper carpunya Ruiz & Pav. (syn Piper lenticellosum C.D.C.) (Piperaceae), are widely used in folk medicine in tropical and subtropical countries of South America as an anti-inflammatory, anti-ulcer, anti-diarrheal and anti-parasitical remedy as well as an ailment for skin irritations., Aims of the Study: To study the anti-inflammatory, anti-secretory and anti-Helicobacter pylori activities of different fractions isolated from an ethanolic extract of the leaves of Piper carpunya, in order to provide evidence for the use of this plant as an anti-ulcer remedy. Moreover, to isolate the main compounds of the extract and relate their biological activity to the experimental results obtained with the fractions., Materials and Methods: Sixteen fractions were obtained from the ethanolic extract (F I-XVI) and 16 pure compounds were isolated and identified from these fractions. We studied the effects of the fractions (0.1-400microg/mL) on the release of myeloperoxidase (MPO) enzyme from rat peritoneal leukocytes, on rabbit gastric microsomal H(+), K(+)-ATPase activity and anti-Helicobacter pylori anti-microbial activity using the microdilution method (MM). The main compounds contained in the fractions were isolated and identified by (1)H- and (13)C NMR spectra analysis and comparison with the literature data., Results: Eight fractions showed inhibition of MPO enzyme (F I-IV, X, XII, XIV and XV). The highest inhibition was observed with F XIV (50microg/mL, 60.9%, p<0.001). F X and XII were the most active ones, inhibiting the gastric H(+), K(+)-ATPase activity with IC(50) values equal to 22.3microg/mL and 28.1microg/mL, respectively. All fractions, except F XV, presented detectable anti-Helicobacter pylori activity, with a diameter of inhibition zones ranging from 11mm up to 50mm. The best anti-Helicobacter pylori activity was obtained with F III and V. Both fractions killed Helicobacter pylori with lowest concentration values, about 6.25mug/mL. Sixteen pure compounds were isolated, five of them are flavonoids that possess strong anti-oxidant and free radical scavenging activity, e.g. vitexin, isovitexin, and rhamnopyranosylvitexin. Terpenoids like sitosterol, stigmasterol and phytol, which have shown gastroprotective activity, and dihydrochalcones, like asebogenin, with anti-bacterial activity, were also isolated. Furthermore, the rare neolignan 1, that is a DNA polymerase beta lyase inhibitor, and (6S, 9S)-roseoside, that shows strong anti-bacterial activity, were isolated, for the first time, from the genus Piper., Conclusions: We suggest that the flavonoids isolated from F I and II (vitexin, isovitexin, rhamnopyranosylvitexin and isoembigenin) contribute to the anti-MPO activity, as well as to their anti-Helicobacter pylori activity. These flavonoids may also be responsible for the important inhibition of H(+), K(+)-ATPase activity. Also the phytosterols and phytol obtained from F XIV and XV could be involved in these gastroprotective activities. These results encourage us to continue phytochemical studies on these fractions in order to obtain full scientific validation for this species., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Transcripts encoding for alpha1, beta1, delta, gamma and epsilon (and its splice variant epsilon(s)) subunits of the muscle-type nicotinic acetylcholine receptor (nAChR) were assessed using reverse transcription followed by polymerase chain reaction (RT-PCR) assays, with RNA extracted from the mouse skeletal muscle (diaphragm) and brain regions (cortex, hippocampus and cerebellum). The presence of alpha1, beta1, delta, gamma, epsilon and epsilon(s) transcripts was confirmed in the diaphragm muscle, used as positive control. mRNAs coding for muscle alpha1, beta1, delta, epsilon, epsilon(s), but not gamma subunits, were detected in adult mouse brain regions. An epsilon-subunit sequence variant, named epsilon(t), was also detected in all brain regions examined, but not in skeletal muscle. This new epsilon-subunit splice variant lacks a 115 bp cassette corresponding to exon 8 in the first intracellular transmembrane domain of the subunit, leading to a truncated protein. The data provide evidence for the presence of muscle-type nAChR subunits in the mouse central nervous system., (Copyright 2009 Elsevier Inc. All rights reserved.)

New compounds that target nicotinic receptors (nAChRs) have been sought to correct disorders affecting cholinergic transmission in central and peripheral synapses. A quaternary derivate of l-hyoscyamine, phenthonium (Phen), was shown by our group to enhance the spontaneous acetylcholine (ACh) release without altering the nerve-induced transmitter release at the neuromuscular junction. The effect was unrelated to membrane depolarization, and was not induced by an increase of calcium influx into the nerve terminal. Phen also presented a competitive antimuscarinic activity and blocked noncompetitively the neuromuscular transmission. In this work we re-examined the mechanisms underlying the facilitatory actions of Phen on [(3)H]-ACh release in isolated ganglia of the guinea pig ileal myenteric plexus. Exposure of the preparations to Phen (10-50 microM) increased the release of [(3)H]-ACh by 81 to 68% over the basal. The effect was not affected by the ganglionic nAChR antagonist hexamethonium (1 nM) at a concentration that inhibited the increase of [(3)H]-ACh release induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP, 30 microM). Association of Phen (10 microM) with DMPP potentiated the facilitatory effect of Phen. [(3)H]-ACh release was not altered by the muscarinic antagonists atropine (1 nM) or pirenzepine (1 microM). However, both antagonists inhibited the release of [(3)H]-ACh induced by either the muscarinic M1 agonist McN-343 (10 microM) or Phen (20 microM). The facilitatory effect of Phen was not altered by CdCl(2) (50 mM), but it was potentiated in the presence of tetraethylammonium (40 mM). The results indicate that the facilitatory action of Phen appears to be mediated by an increase of the inwardly rectifying potassium channels conductance probably related to the compound antimuscarinic activity.

Phenthonium (Phen), a quaternary analog of hyoscyamine, is a blocker of muscarinic activity and an allosteric blocker of alpha(1)2betagammaepsilon nicotinic receptors. Specifically, Phenthonium increases the spontaneous release of acetylcholine at the motor endplate without depolarizing the muscle or inhibiting cholinesterase activity. This paper compares Phenthonium's effects on sympathetic transmission and on ganglionic nicotinic receptor activation. Neurotransmitter release and twitch of the rat vas deferens were induced either by electrical stimulation or by 1,1-dimethyl-4-phenylpiperazine (DMPP) activation of nicotinic receptors. Contractions independent of transmitter release were induced by noradrenaline and adenosine 5'-triphosphate (ATP). Phenthonium inhibited transmitter release and depressed twitch without changing the responsiveness to noradrenaline or ATP. Twitch depression did not occur after K(+)-channel blockade with 4-aminopyridine (4-AP) or charybdotoxin. DMPP had a similar effect, but high concentrations induced contraction of non-stimulated organs. Incubation of Phenthonium inhibited further DMPP twitch depression and non-competitively depressed the contractile responses elicited by DMPP. Furthermore, mecamylamine, but neither methyllycaconitine nor atropine, blocked the contraction elicited by DMPP. Phenthonium and DMPP are K(+)-channel openers that primarily inhibit sympathetic transmission. Contraction induced by DMPP was probably mediated by neuronal nicotinic receptor other than the alpha7 subtype. The blockade of DMPP contractile response was unrelated to Phenthonium's antimuscarinic or K(+)-channel opening activities. Since Phenthonium's quaternary chemical structure limits its membrane diffusion, the non-competitive inhibition of DMPP excitatory responses should be linked to allosteric interaction with neuronal nicotinic receptors that putatively qualify Phenthonium as a novel modulator of cholinergic synapses.

To analyse the effect of ageing on the projection of the anterior interposed nucleus to the red nucleus, we injected the retrograde tracer fluorogold in the red nucleus of 3-, 6- and 12-month-old mice. The number of labelled neurones in the anterior interposed nucleus fell by 9% between 3 and 6 months and by another 9% between 6 and 12 months (all P < 0.001). This suggests that loss of neurones from the cerebellar nuclei starts well before old age.

The absence of dystrophin in Duchenne muscular dystrophy (DMD) and in the mutant mdx mouse causes muscle degeneration and disruption of the neuromuscular junction. Based on evidence from the denervation-like properties of these muscles, we assessed the ligand-binding constants of nicotinic acetylcholine receptors (nAChRs) and the mRNA expression of individual subunits in membrane preparations of diaphragm muscles from adult (4-month-old) and aged (20-month-old) control and mdx mice. The concentration of nAChRs as determined by the maximal specific [(125)I]-alpha-bungarotoxin binding (Bmax) in the muscle membranes did not change with aging in both animal strains. When compared to age-matched control groups, the Bmax in mdx muscles was increased by 65% in adults, and by 103% in aged mice with no alteration of toxin affinity for nAChRs. Reverse-transcription polymerase chain reaction assays showed that mRNA transcripts for the nAChR alpha1, gamma, alpha7, and beta2, but not the epsilon subunits, were more abundant in mdx than in control muscles. The results indicate increased expression of extrajunctional nAChRs in the mdx diaphragm and reflect impairment of nAChR regulation in dystrophin-deficient muscles. These observations may be related to the resistance to nondepolarizing muscle relaxants and the high sensitivity to depolarizing agents reported in DMD patients.

Lack of dystrophin is known to reduce several cerebral fiber systems. To investigate if the loss of fibers is progressive, we analyzed projections of the trigeminal sensory system to the red nucleus in 3, 6, and 12 month old dystrophin-deficient mdx mice. The retrograde tracer fluorogold was injected in the magnocellular part of the red nucleus, and the number of labeled neurons in the oral part of the spinal trigeminal nucleus (Sp5O) was counted. We found that the number of labeled Sp5O neurons was reduced by 50% in mdx mice compared to age-matched control mice. The number of labeled Sp5O neurons did not change significantly between 3 and 12 months neither in mdx nor in control mice. In addition, the number of labeled neurons in the interstitial system of the trigeminal nerve was reduced by 43% in mdx mice. We conclude that fiber loss did not continue beyond the age of 3 months. Our data suggest that lack of full-length dystrophin impairs neuronal migration or axonal outgrowth, or increases neuronal death during fetal or early life.

Cecropia glazioui Sneth (Cecropiaceae) is used in folk medicine in tropical and subtropical Latin America as cardiotonic, diuretic, hypotensive, anti-inflammatory and anti-asthmatic. The hypotensive/antihypertensive activity of the plant aqueous extract (AE) and isolated butanolic fraction (BuF) has been confirmed and putatively related to calcium channels blockade in vascular smooth musculature [Lapa, A.J., Lima-Landman, M.T.R., Cysneiros, R.M, Borges, A.C.R., Souccar, C., Barreta, I.P., Lima, T.C.M., 1999. The Brazilian folk medicine program to validate medicinal plants - a topic in new antihypertensive drug research. In: Hostettman, K., Gupta, M.P., Marston, A. (Eds.), Proceedings Volume, IOCD/CYTED Symposium, Panamá City, Panamá, 23-26 February 1997. Chemistry, Biological and Pharmacological Properties of Medicinal Plants from the Americas. Harwood Academic Publishers, Amsterdam, pp. 185-196; Lima-Landman, M.T., Borges, A.C., Cysneiros, R.M., De Lima, T.C., Souccar, C., Lapa, A.J., 2007. Antihypertensive effect of a standardized aqueous extract of Cecropia glaziovii Sneth in rats: an in vivo approach to the hypotensive mechanism. Phytomedicine 14, 314-320]. Bronchodilation and antidepressant-like activities of both AE and BuF have been also shown [Delarcina, S., Lima-Landman, M.T., Souccar, C., Cysneiros, R.M., Tanae, M.M., Lapa, A.J., 2007. Inhibition of histamine-induced bronchospasm in guinea pigs treated with Cecropia glaziovi Sneth and correlation with the in vitro activity in tracheal muscles. Phytomedicine 14, 328-332; Rocha, F.F., Lima-Landman, M.T., Souccar, C., Tanae, M.M., De Lima, T.C., Lapa, A.J., 2007. Antidepressant-like effect of Cecropia glazioui Sneth and its constituents -in vivo and in vitro characterization of the underlying mechanism. Phytomedicine 14, 396-402]. This study reports the antiulcer and antisecretory gastric acid activities of the plant AE, its BuF and isolated compounds with the possible mechanism involved. Both AE and BuF were assayed on gastric acid secretion of pylorus-ligated mice, on acute models of gastric mucosal lesions, and on rabbit gastric H(+), K(+)-ATPase preparations. Intraduodenal injection of AE or BuF (0.5-2.0g/kg, i.d) produced a dose-related decrease of the basal gastric acid secretion in 4-h pylorus-ligated mice. At 1.0g/kg, BuF decreased the volume (28%) and total acidity (33%) of the basal acid secretion, and reversed the histamine (2.5mg/kg, s.c.)- or bethanecol (1.0mg/kg, s.c.)-induced acid secretion to basal values, indicating inhibition of the gastric proton pump. Pretreatment of mice with the BuF (0.05-0.5g/kg, p.o.) protected against gastric mucosal lesions induced by 75% ethanol, indomethacin (30mg/kg, s.c.) or restraint at 4 degrees C. BuF also decreased the gastric H(+), K(+)-ATPase activity in vitro proportionately to the concentration (IC(50)=58.8microg/ml). The compounds isolated from BuF, consisting mainly of cathechins, procyanidins and flavonoids [Tanae, M.M., Lima-Landman, M.T.R., De Lima, T.C.M., Souccar, C., Lapa, A.J., 2007. Chemical standardization of the aqueous extract of Cecropia glaziovii Sneth endowed with antihypertensive, bronchodilator, antacid secretion and antidepressant-like activities. Phytomedicine 14, 309-313], inhibited the in vitro gastric H(+), K(+)-ATPase activity at equieffective concentrations to that of BuF. The results indicate that C. glazioui constituents inhibit the gastric proton pump; this effect may account for the effective antisecretory and antiulcer activities of the standardized plant extract.

We studied projections from the interstitial system of the spinal trigeminal tract (InSy-S5T) to the red nucleus of the mouse with retrograde tracers (fluorogold and latex microbeads impregnated with rhodamine and fluorescein). Injections in the magnocellular part of the red nucleus caused labeling of cells in the rostral, intermediate, and caudal paratrigeminal nucleus (Pa5), dorsal paramarginal nucleus (PaMD), insular trigemeo-lateral cuneate nucleus (I5CuL), and the trigeminal extension of the parvocellular reticular formation (5RPC). All projections were bilateral, but contralateral projections were stronger. The number of retrogradely labeled cells in the InSy-S5T in 3-, 6-, and 12-month-old mice was similar. Injections restricted to the parvocellular red nucleus did not label the nuclei of the InSy-S5T. This projection from the InSy-S5T to the red nucleus may mediate modulation of the facial muscles by pain and other sensory information.

The present study aimed to characterize the antidepressant-like effect of a standardized aqueous extract (AE) of Cecropia glazioui Sneth and its purified fractions on in vivo (forced swimming test), ex vivo (hippocampal monoamines levels) and in vitro (serotonin, noradrenaline and dopamine uptake) tests, searching for the active principles and the underlying mechanisms of action. Treatment with AE, or with its butanolic fraction (BuF), the latter rich in catechins, procyanidins and flavonoids, reduced the immobility of rats in the forced swimming test indicating an antidepressant-like effect. Biochemical analysis of the hippocampal neurotransmitters in BuF-treated rats showed significant increase in monoamines levels. BuF and six of its purified constituents inhibited the uptake of [(3)H]-serotonin, [(3)H]-dopamine and [(3)H]-noradrenaline by synaptosomes of different brain regions. Catechin, catechin (4alpha-->8) ent-catechin (Procyanidin B3 isomer) and epicatechin (4beta-->8) epicatechin (Procyanidin B2) were the most active compounds. Comparatively, the uptake of [(3)H]-noradrenaline was the most affected. These results show that the antidepressant-like effect promoted by C. glazioui extract is most likely due to the blockade of the monoamines uptake in the CNS.

The freeze-dried aqueous extract (AE) from the aerial parts of Scoparia dulcis was tested for its effects on experimental gastric hypersecretion and ulcer in rodents. Administration of AE to animals with 4h pylorus ligature potently reduced the gastric secretion with ED(50)s of 195 mg/kg (rats) and 306 mg/kg (mice). The AE also inhibited the histamine- or bethanechol-stimulated gastric secretion in pylorus-ligated mice with similar potency suggesting inhibition of the proton pump. Bio-guided purification of the AE yielded a flavonoid-rich fraction (BuF), with a specific activity 4-8 times higher than the AE in the pylorus ligature model. BuF also inhibited the hydrolysis of ATP by H(+),K(+)-ATPase with an IC(50) of 500 microg/ml, indicating that the inhibition of gastric acid secretion of Scoparia dulcis is related to the inhibition of the proton pump. Furthermore, the AE inhibited the establishment of acute gastric lesions induced in rats by indomethacin (ED(50)=313 mg/kg, p.o.) and ethanol (ED(50)=490 mg/kg, p.o.). No influence of the AE on gastrointestinal transit allowed discarding a possible CNS or a cholinergic interaction in the inhibition of gastric secretion by the AE. Collectively, the present data pharmacologically validates the popular use of Scoparia dulcis in gastric disturbances.

This study reports the extraction process and standardization of the aqueous extract (AE) of a Cecropia species aiming its pharmacological characterization as a phytomedicine to be used in primary health care. The plant was originally collected in its environment, and was thereafter specially cultivated for the present work. To standardize the plant AE, several 2.0% tea of the dried leaves were prepared under controlled conditions and freeze dried. The AE (20% yield) was partitioned with n-butanol yielding the butanolic fraction (BuF; 1% yield). The activity of AE on vital organ functions (cardiovascular, respiratory, gastrointestinal and central nervous system) was determined in vivo. The effects of AE were compared to those of BuF in the same models and the relative potency determined. BuF was further evaluated in representative in vitro models to assess possible mechanisms of action. Chemical constituents of BuF were isolated in preparative HPLC columns yielding 10 highly purified compounds chemically identified as catechins (2), procyanidins (4), flavonoids (2), mixed sugars (1) and chlorogenic acid. All the compounds were identified by chemical analytic instrumentation (13C-NMR, 1H-NMR, LC-MS). Their relative concentrations in AE were ca 12% catechins, 19% procyanidins and 19% flavonoids. The pharmacological activity of the standardized AE is reported in accompanying papers.

Cecropia glaziovii Sneth is a common tree at the Southeastern Brazilian coast. As many other species of the genus, it shares the reputed folk use to treat heart failure, cough, asthma and bronchitis. The plant has been cultivated under controlled conditions and the 2% aqueous extract (AE) prepared with the dried leaves was standardized by its chemical contents on catechins, flavonoids and procyanidins. The present paper reports the antihypertensive activity of AE and of n-butanol fraction (BuF), an enriched semi-purified butanolic fraction used to isolate the main chemical constituents. Oral administration of AE and BuF induced hypotension in normotensive rats. The effect of AE (0.5 g/kg/bi, p.o.) was time and dose-dependent peaking at 2-3 weeks after daily administration. BuF was faster but not more active than AE. Both extracts decreased the hypertension of spontaneous hypertensive rats, the hypertension induced in rats by L-NAME treatment and that induced by constriction of one renal artery. The antihypertensive effect was maintained for as long as 60 days of treatment and was reversible upon drug washout at the same rate of its establishment. Acute i.v. administration of BuF to anesthetized rats induced a fast short-lasting hypotension and inhibited the pressor responses to noradrenaline, angiotensin I and angiotensin II by 40%. These results were indirect indications that the hypotension induced by AE is not related to ACE inhibition, increased NO synthesis, or specific blockade of alpha1 and AT1 receptors. It can be suggested that BuF interferes with the calcium handling mechanisms in smooth muscle cells and neurons. Intravenous injection of five out of nine compounds isolated from BuF produced immediate but short-lasting hypotension that does not correlate with the onset of the hypotension after oral treatment. This finding suggests that they may not be the compounds directly responsible for the delayed and sustained hypotension after per os administration of AE. The many compounds isolated from AE are under evaluation to determine its pharmacokinetics, mechanisms of action and interactions necessary to yield the plant effect. Although its mechanism is still unknown, AE seems to be an effective and safe antihypertensive phytomedicine.

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8+/-2.6 nmol/min/ml), intermediate ACE (39.4+/-1.4 nmol/min/ml) and low ACE (23.5+/-0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7+/-0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.

A standardized aqueous extract (AE) and a purified fraction (BuF) of Cecropia glaziovi Sneth leaves were tested in unrestrained guinea pigs challenged with histamine. Changes of the respiratory pressure and rate were recorded in a whole body plethysmograph before and after treatment. The concentration of histamine necessary to produce bronchospasm was increased by five-fold following administration of AE (1.0 g/kg p.o.), and by two-fold after treatment with the semi-purified procyanidin/flavonoids enriched BuF (0.1 g/kg p.o.). Both effects were blocked by previous treatment with propranolol (10.0 mg/kg i.p.). In vitro incubation of BuF (0.1-1.0 mg/ml) decreased by 13-55% the maximal response of guinea pig tracheal muscle to histamine, without significant change of EC50. The results confirmed old reports on the useful pulmonary effects of Cecropia extracts. The bronchodilation observed in vivo seems to be related to beta-adrenergic activity observed in vitro only with high concentrations of the purified extract.

This work assessed the mechanism underlying the antisecretory gastric acid effect of Plectranthus barbatus Andrews (Lamiaceae) and active constituents. Popularly known as "false-boldo", this plant is used in Brazilian folk medicine to treat gastrointestinal and hepatic ailments. The plant aqueous extract (AE) and isolated compounds were assayed in vivo in pylorus-ligated mice, and in vitro on acid secretion measured as [(14)C]-aminopyrine ([(14)C]-AP) accumulation in rabbit gastric glands and gastric H(+),K(+)-ATPase preparations. Injected into the duodenal lumen, the AE of the plant leaves (0.5 and 1.0 g/kg) decreased the volume (62 and 76%) and total acidity (23 and 50%) of gastric acid secretion in pylorus-ligated mice. Bioguided purification of the AE yielded an active fraction (IC(50)=24 microg/ml) that inhibited acid secretion in rabbit gastric glands with a potency 10 to 18 times greater than that of the originating extract, on both the basal and stimulated acid secretion by histamine (His) (1 microM) or bethanechol (100 microM). At the same concentrations the gastric H(+),K(+)-ATPase activity was also inhibited. The active constituent was chemically identified as the abietanoid dienedione plectrinone A which reduced the H(+),K(+)-ATPase activity with IC(50)=171 microM. The results indicate that inhibition of the gastric proton pump by this diterpenoid may account for the antisecretory acid effect and reputed anti ulcer activity of Plectranthus barbatus.

The lack of dystrophin that causes Duchenne muscle disease affects not only the muscles but also the central nervous system. Dystrophin-deficient mdx mice present changes in several brain fiber systems. We compared the projections from the trigeminal sensory nuclear complex to the red nucleus in control and mdx mice using retrograde tracers. Injection of 200 nL 2% fluorogold into the red nucleus caused labeling in the mesencephalic trigeminal nucleus, the principal sensory nucleus and the oral, interpolar, and caudal subnuclei of the spinal trigeminal nucleus in both control and mdx mice. Injection of latex microbeads labeled with rhodamine and fluorescein gave results similar to those seen with fluorogold. The number of labeled neurons in the trigeminal sensory nuclear complex was significantly reduced in mdx mice. In the oral subnucleus of the spinal trigeminal nucleus this reduction was 50%. These results indicate that the trigemino-rubral pathway is reduced in dystrophin-deficient mice.

Clibadium surinamense L, popularly known as cunambi, is a native plant from the Northern region of Brazil illegally used for predatory fishing. Previous results from our laboratory have demonstrated that the oral treatment of mice with the ethanolic extract (EE) of the leaves of the plant induced generalized tonic-clonic seizures followed by death within 30 min. The aims of the present paper were to characterize the convulsant effect of the hexanic extract (HE) of the stems and leaves of C. surinamense and, by bioguided purification, to identify the active principle and its mechanism of action. The leaves and stems were extracted with hexane (100 g/L) in Soxhlet for 36 h (yield of 2.4%), the solvent was evaporated and the powder dissolved in 1.5% saline/Tween 80. Male mice (30-35 g) treated with HE (22.5-360 mg/kg, p.o.) showed behavioral alterations consistent with CNS stimulation. The intensity and duration of the effect were proportional to the administered doses. The behavioral alterations, measured with a graded score of seizure severity, revealed that pretreatment with carbamazepine (30 mg/kg, i.p., 60 min) or phenytoin (50 mg/kg, i.p., 30 min) did not alter the HE convulsive effect. In contrast, phenobarbital (30 mg/kg, i.p., 60 min) or diazepam (2 mg/kg, i.p., 30 min) reduced the HE effect, increasing the ED(50) for clonic seizures from 64.4 to 89.8 mg/kg and 168.9 mg/kg, respectively. Purification of the HE in a silica gel column eluted with a hexane/ethyl acetate gradient yielded a single fraction with convulsant effect in which cunaniol acetate was identified by (1)H NMR as the main active compound. These results indicated that inhibition of GABAergic transmission by cunaniol acetate might be responsible for the convulsant effects of C. surinamense L in mice, but do not exclude a direct cunaniol action labilizing neuronal excitability.

The time course of muscular dystrophy on the androgen-sensitive levator ani muscle was compared to that of the diaphragm of dystrophic (mdx) mice aged 1-20 months. Muscle growth, isometric contractile properties and caffeine-induced contractures were determined to assess the hormone myotrophic effect, muscle strength and sarcoplasmic reticulum function, respectively, of both control and dystrophic muscles. Histological analysis of mdx muscles showed variable fiber size, centronucleated cells, infiltration of connective tissue, and necrosis which was less severe in the levator ani than in the diaphragm muscle. Tetanic tension per unit weight in the mdx levator ani was reduced (29%) after aging, while the contraction time remained unchanged. The tetanic tension of the mdx diaphragm muscle decreased with age from 3 to 20 months (20-64%), and the relaxation time was prolonged after aging (22%). Gonadectomy of young adult mdx mice caused atrophy of the levator ani muscle, accelerated muscle wasting, reduced the tetanic force (31%), but it did not affect the contraction time and caffeine responses. The results showed that testosterone does not prevent the progress of muscle disease in the mdx levator ani, but androgen withdrawal accelerates muscle wasting suggesting a normonal beneficial effect.

Accidents caused by the venomous fish Thalassophryne nattereri are characterized by edema, intense pain and necrosis at the site of the sting. This study assessed the nociceptive and edematogenic activities of T. nattereri venom after injection into the mouse hindpaw and determination of the paw licking duration and weight. Subplantar injections of the venom (0.1-6 microg) induced a dose-related increase of the paw licking time and paw swelling with maximal values at 3 microg (209.5 +/- 57.5 s and 135.0 +/- 6.8 mg, respectively). Pretreatment of mice with either indomethacin (10 mg/kg, i.p.), a cyclooxygenase inhibitor, dexamethasone (1 mg/kg, s.c.), a steroid anti-inflammatory agent, cyproheptadine (1 mg/kg, i.p.), antagonist of serotonin receptors or L-NAME (100 mg/kg, s.c.), inhibitor of nitric oxide syntase, did not affect the venom-induced nociceptive and edematogenic responses. Injection of the opioid analgesic fentanyl (0.1 mg/kg, s.c.) reduced the paw licking time induced by 1 microg venom by 84% of control, without affecting the paw swelling. Both nociceptive and edematogenic responses were reduced after treatment with a specific tissue kallikrein inhibitor (TKI, 100 mg/kg, i.p.) by 78% and 24% from control values, respectively. Administration of a specific plasma kallikrein inhibitor (PKSI(527,) 100 mg/kg, s.c.) did not affect the venom-induced nociceptive response, but it decreased the paw edema by 15% from control. After injection of the angiotensin-converting enzyme inhibitor captopril (100 mg/kg, i.p.) the venom-induced nociceptive end edematogenic responses were increased by two-fold. The role of kallikreins possibly present in the venom was further assessed by hydrolysis of human kininogen and kininogen-derived synthetic peptides, showing the release of kallidin (Lys-bradykinin). The hydrolysis was inhibited by metal chelating agents but not by serino-, aspartyl- or cysteino-proteinase inhibitors. The data suggest that a protease with tissue-kallikrein-like activity plays a major role in nociception and edema induced by T. nattereri venom and this should be considered to achieve efficient treatments for human accidents with this venom.

Freeze-dried aqueous extracts (AEs, 0.1-1g/kg body wt., p.o.) obtained from entire or selected parts of Stachytarpheta cayennensis were tested for their effects on gastric secretion, gastric motility, inflammation and pain in rodents, with the purpose of validating the plant's ethnomedical uses. The AE-Total, AE-Flowers and AE-Leaves but not AE-Stems inhibited the gastric acid secretion in pylorus-ligated rats with varying potency. Purification of AEs yielded the semipurifed fractions EtFs rich in iridoids. All the EtFs with exception of EtF-Stems inhibited gastric acid secretion of pylorus ligated mice. While AE-Total stimulated the intestinal transit of mice by 43%, AE-Leaves delayed it by 38%. These effects on intestinal transit were not observed when the EtFs were tested. Only AE-Leaves and AE-Flowers altered the gastric emptying of semisolids, increasing it by 45% and 69%, respectively. These results indicate that the compounds related to inhibition of gastric acid secretion and gastrointestinal motility are different. The AE-Total reduced abdominal writhing induced by acetic acid potently (ED50 value = 700 mg/kg, p. o.) without altering the writhes induced by acetylcholine. Attempts to identify the mechanism of analgesia were unsuccessful since the AE-Total did not show analgesic effects when tested in different models of pain such as formalin and capsaicin or the tail-flick test. Pretreatment of animals with AE-Total did not show antiinflammatory activity in any of the acute (paw edema induced by carrageenin, dextran or histamine, pleurisy induced by carrageenin and capsaicin-induced mouse ear edema) or chronic (air pouch) models used. No toxic signs were observed after administration of the different extracts up to 2 g/kg body wt., p.o. Collectively, the results confirmed folk information indicating presence of analgesic, mild laxative and potent inhibition of gastric secretion activities in the aqueous extracts of S. cayennensis. The results do not, however confirm the folk use of the plant as an antiinflammatory medicine.

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 micro g/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 micro g/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 micro M acetylcholine alone and cumulative concentrations of 1 micro M to 10 mM were unaffected. At venom concentrations higher than 50 micro g/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24 degrees C, the venom (50 g/ml) produced only partial neuromuscular blockade (30.7 +/- 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.

Solanum paniculatum L. is used commonly in Brazilian folk medicine for the treatment of liver and gastrointestinal disorders. The freeze-dried aqueous extracts (WEs) obtained from distinct parts of the plant (flowers, fruits, leaves, stems and roots) were tested to determine their antiulcer and antisecretory gastric acid activities using mice. The aqueous extracts of roots, stems and flowers inhibited gastric acid secretion in pylorus-ligated mice with ED50 values of 418, 777 and 820 mg/kg body wt. (i.d.), respectively. Extracts of leaves (0.5-2 g/kg body wt., i.d.) did not affect gastric secretion, whereas fruit extracts (0.5-2 g/kg body wt., i.d.) stimulated gastric acid secretion. The stimulatory effect of the fruit extract was inhibited by pretreatment with atropine (5 mg/kg body wt., i.m.) but not with ranitidine (80 mg/kg body wt., i.p.) suggesting that the fruit extract activates the muscarinic pathway of gastric acid secretion. In contrast, administration of the root extract into the duodenal lumen inhibited histamine- and bethanechol-induced gastric secretion in pylorus-ligated mice. In addition, the aqueous extract of roots (ED50 value, 1.2 g/kg body wt., p.o.) protected the animals against production of gastric lesions subsequent to the hypersecretion induced in mice by stress following cold restraint. This effect was not reproduced when the lesions were induced by blockade of prostaglandins synthesis via subcutaneous injection of indomethacin. Thus, antiulcer activity of the plant extracts appears to be related directly to a potent anti-secretory activity. No toxic signs were observed following administration of different extracts up to 2 g/kg body wt., p.o. Collectively, the results validate folk use of Solanum paniculatum L. plant to treat gastric disorders.