A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.

INTRODUCTION: The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone). OBJECTIVE: To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers. METHODOLOGY: The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C18-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)3NH2 (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min. RESULTS: The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits. CONCLUSION: The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.

Neolignans from plants in northeastern Brazil (Lauraceae) with activity against Trypanosoma cruzi.

Trypanosoma cruzi is the ethiological agent for Chagas disease in Latin America. This study aimed to test the trypanocidal effect of licarin A and burchellin isolated from plants in northeastern Brazil. These neolignans were tested on T. cruzi and on peritoneal macrophages, to evaluate drug toxicity. Epimastigote growth was inhibited in 45% with licarin A and 20% with burchellin with an IC(50)/96 h of 462.7 microM and 756 microM, respectively. Epimastigotes treated with licarin A presented swollen mitochondria and disorganized mitochondrial cristae, kDNA and Golgi complex. When treated with burchellin, they presented enormous autophagosomes and chromatin disorganization. Licarin A and burchellin were able to induce trypomastigote death with IC(50)/24 h of 960 microM and 520 microM, respectively. Although licarin A presented an IC(50) for trypomastigotes higher than for epimastigotes, both substances acted as therapeutic trypanocidal agents, because they were able to kill parasites without affecting macrophages. Due to our results, burchellin and licarin A need to be further analysed to observe if they may be used as alternative blood additive prophylaxis against Chagas disease, since it has been established that blood transfusion is an important mechanism in the transmission process.

In vivo effect of essential oil of Mentha x villosa and its active compound against Schistosoma mansoni (Sambon, 1907).

Schistosomiasis treatment is dependent on a single drug, praziquantel (PZQ). The development of resistance of PZQ has drawn the attention of many researchers to alternative drugs. One viable and promising treatment is the study of medicinal plants as a new approach to the experimental treatment for Schistosomiasis. The present work aimed to evaluate in vivo antischistosomal activity of effect of Mentha x villosa Oil Essential (Mv-EO) and rotundifolone (ROT) against Schistosoma mansoni. Thirty-day-old female Swiss webster mice (Mus musculus) weighing 50 grams were used. Mice were infected with 80 cercariae of S. mansoni (BH strain) and orally administered Mv-EO (50, 100 and 200 mg/Kg) and ROT (35.9, 70.9 and 141.9 mg/Kg) at 45-days post infection for 5 consecutive days. All mice were euthanized 60 days after infection. Praziquantel was the positive control in the experiment. Doses of 200 mg/kg (Mv-EO) and ROT (141.9 mg/Kg) resulted in a significant reduction in fluke burden (72.44% and 74.48%, respectively). There was also marked reduction in liver, intestinal and faecal and changed oogram pattern, compared to infected untreated mice. Considering the results obtained, further biological studies are required in order to elucidate the mechanism of schistosomicidal action on against adult S. mansoni.

Warifteine, a bisbenzylisoquinoline alkaloid, decreases immediate allergic and thermal hyperalgesic reactions in sensitized animals.

Warifteine is a bisbenzylisoquinoline alkaloid isolated from the Cissampelos sympodialis Eichl (Menispermaceae). This plant is used in the folk medicine for the treatment of airway respiratory diseases. A murine model of immediate allergic reaction was used to evaluate warifteine treatment in the IgE production, leukocyte activation, thermal hyperalgesia, mast cell degranulation and scratching behavior. BALB/c mice treated with warifteine (0.4-10 mg/Kg) 1 h before OVA sensitization reduced OVA induced paw edema as well as the OVA-specific IgE serum titers as compared with non-treated and OVA-sensitized animals. Warifteine also reduced the mice death evoked by IgE-dependent anaphylactic shock reaction at 30 min after intravenous OVA challenge. To assess the effect of warifteine treatment on T cell proliferative response, spleen cells from warifteine treated or non-treated and OVA-sensitized animals were evaluated. Spleen cells from warifteine treated animals (2.0 mg/kg) did not proliferate following OVA stimulation as compared with spleen cell cultures from non-treated animals. This response may be related with the increase of NO production as observed in peritoneal macrophage cultures treated with warifteine. Thermal hyperalgesia evoked by IgE or histamine/5-hydroxytryptamine challenge was inhibited on rats at dose of 4.0 mg/kg. Warifteine treatment (0.6 or 6.0 microg/ml) also decreased the IgEalphaDNP-BSA sensitized mast degranulation after DNP-BSA challenge measured by histamine release. In addition, compound 48/80-induced scratching behavior was also sensitive to warifteine treatment. These results demonstrate for the first time that warifteine treatment reduced the allergy-associated responses.

The triterpenoid lupeol attenuates allergic airway inflammation in a murine model.

Asthma is a chronic inflammatory disease of the airways associated with a Th2 immune response. Despite their side effects, corticosteroids are the most used and effective drugs for treatment of asthma. In this work we investigated the efficacy of lupeol, a triterpenoid isolated from Lonchocarpus araripensis [corrected] Benth. (Fabaceae), in the treatment of bronchial asthma in BALB/c mice immunized with ovalbumin. Administration of lupeol caused the reduction of cellularity and eosinophils in the bronchoalveolar lavage fluid. Treatment with lupeol also reduced the production of mucus and overall inflammation in the lung. Levels of Type II cytokines IL-4, IL-5 and IL-13 were significantly reduced in mice treated with lupeol, an effect that was similar to that observed in dexamethasone-treated mice. In contrast, IgE production was not significantly altered after treatment with lupeol. In conclusion, our results demonstrate that lupeol attenuates the alterations' characteristics of allergic airway inflammation. The investigation of the mechanisms of action of this molecule may contribute for the development of new drugs for the treatment of asthma.

Biological activity of neolignans on the post-embryonic development of Chrysomya megacephala.

Phytochemicals endowed with hormonal, antihormonal, or toxic activity are potential agents for insect control. The effect of some neolignans on one of the most prevalent flies in urban areas, which constitutes a menace to public health, was investigated.

Acute intoxication by Crotalaria retusa in sheep.

Acute intoxication by seeding Crotalaria retusa occurred during the dry season, in a flock of 80 sheep, in the semi-arid region of the state of Paraiba, northeastern Brazil. Anorexia, severe depression, mild jaundice, incoordination and recumbence were observed in 16 sheep that died within 12 h. At necropsy the liver had a nutmeg appearance. Seeds of C. retusa were found in large amounts in the rumen of the dead animals. Histologic lesions of the liver were characterized by centrilobular necrosis. Seeds of C. retusa were given to six sheep at doses of 2.5 (two sheep), 5, 10, 20 and 40 g/kg bw (one sheep for each dose). Sheep ingesting 5-40 g/kg bw were killed when moribund, 38-120 h after the start of the administration. Clinical signs and gross and histologic lesions were similar to those observed in field outbreak. The seeds used in the experiment contained 1.4% of monocrotaline. It is concluded that hungry sheep can be affected spontaneously by acute monocrotaline intoxication when they ingest large amounts of C. retusa seeds in a short period of time.

In vivo effect of essential oil of Mentha x villosa and its active compound against Schistosoma mansoni (Sambon, 1907) / Efeito in vivo do óleo essencial de Mentha x villosa e seu composto ativo sobre Schistosoma mansoni (Sambon, 1907)

Abstract Schistosomiasis treatment is dependent on a single drug, praziquantel (PZQ). The development of resistance of PZQ has drawn the attention of many researchers to alternative drugs. One viable and promising treatment is the study of medicinal plants as a new approach to the experimental treatment for Schistosomiasis. The present work aimed to evaluate in vivo antischistosomal activity of effect of Mentha x villosa Oil Essential (Mv-EO) and rotundifolone (ROT) against Schistosoma mansoni. Thirty-day-old female Swiss webster mice (Mus musculus) weighing 50 grams were used. Mice were infected with 80 cercariae of S. mansoni (BH strain) and orally administered Mv-EO (50, 100 and 200 mg/Kg) and ROT (35.9, 70.9 and 141.9 mg/Kg) at 45-days post infection for 5 consecutive days. All mice were euthanized 60 days after infection. Praziquantel was the positive control in the experiment. Doses of 200 mg/kg (Mv-EO) and ROT (141.9 mg/Kg) resulted in a significant reduction in fluke burden (72.44% and 74.48%, respectively). There was also marked reduction in liver, intestinal and faecal and changed oogram pattern, compared to infected untreated mice. Considering the results obtained, further biological studies are required in order to elucidate the mechanism of schistosomicidal action on against adult S. mansoni.

Resumo O tratamento da esquistossomose é dependente de uma única droga, praziquantel (PZQ). O desenvolvimento da resistência de PZQ tem atraído atenção de muitos pesquisadores por medicamentos alternativos. Um tratamento viável e promissor é o estudo das plantas medicinais como uma nova abordagem para o tratamento experimental para esquistossomose. O presente trabalho objetivou avaliar a atividade esquistossomicida in vivo óleo essencial de Mentha x villosa (OE-Mv) e rotundifolona (ROT) contra Schistosoma mansoni. Foram utilizados camundongos Swiss webster (Mus musculus) fêmea de trinta dias de idade pesando 50 gramas. Os camundongos foram infectados com 80 cercárias de S. mansoni (cepa BH) e administrado por via oral OE-Mv (50, 100 e 200 mg/Kg) e ROT (35,9, 70,9 e 141,9 mg/Kg) apos 45 dias de infecção durante 5 dias consecutivos. Todos os animais foram eutanasiados 60 dias após a infecção. Praziquantel foi o controle positivo no experimento. O tratamento dos camundongos infectados com doses de 200 mg/kg (OE-Mv) e rotundifolona (141,9 mg/Kg) resultaram em redução significativa dos vermes (72.44% e 74.48%, respectivamente). Foi observado também redução no fígado, intestino e fecal e alteração no padrão do oograma, em comparação aos camundongos infectados e não tratados. Considerando os resultados obtidos, mais estudos biológicos são necessários a fim de elucidar o mecanismo de ação esquistossomicida contra adultos de S. mansoni.

Protective effects of yangambin on cardiovascular hyporeactivity to catecholamines in rats with endotoxin-induced shock.

The protective effects of a new, selective, plant-derived platelet-activating factor (PAF) antagonist, yangambin, on the cardiovascular alterations and mortality due to endotoxic shock were investigated in anaesthetized rats. We also studied the involvement of PAF in the induction of the vascular and cardiac hyporesponsiveness to adrenergic stimulation observed during endotoxaemia. The animals were sensitized to the lethal effects of Escherichia coli lipopolysaccharide (LPS) with D(+)-galactosamine (50 mg/kg, i.v.) 15 min before LPS injection. LPS (3 mg/kg, i.v.) induced a progressive and marked decrease in mean arterial blood pressure from 85+/-4 to 30+/-3 mmHg and a reduction of cardiac output (CO) from 180+/-7 to 37+/-3 ml/min (120 min) accompanied by a maintenance of systemic vascular resistance, suggesting that cardiovascular collapse resulted mainly from myocardial depression. The maximum pressor responses to noradrenaline (0.3-3.0 microg/kg, i.v.) fell from 72+/-9 (control) to 5+/-1 mmHg (LPS) while the CO responses decreased from 81+/-5 to 8+/-3 ml/min. Pre-treatment with yangambin (30 mg/kg, i.v.) or with WEB 2086 (5 mg/kg, i.v.), a reference PAF receptor antagonist, completely prevented the LPS-induced cardiovascular collapse and abolished the sharp reductions of the arterial blood pressure and CO responses to noradrenaline observed during endotoxaemia. Post-treatment with yangambin 90 min after LPS administration did not reverse the arterial hypotension, cardiac failure or cardiovascular hyporesponsiveness to catecholamines. Finally, the acute (150 min) survival rates of endotoxic shock increased from 0% (LPS group) to 100% in the groups pretreated with either yangambin or WEB 2086. The long-term (7-day) survival also increased from 0% (LPS group) to 85% (yangambin pre-treatment group). In conclusion, these data suggest a role for PAF in the pathogenesis of endotoxin-induced vascular and cardiac hyporesponsiveness to catecholamines and confirm its involvement in the complex cascade of multiple mediators released during endotoxic/septic shock. Yangambin proved to be an effective pharmacological agent against cardiovascular collapse and mortality in endotoxin shock.

Anticonvulsant properties of N-salicyloyltryptamine in mice.

A new tryptamine analogue, N-salicyloyltryptamine (STP), a potential central nervous system (CNS) depressant, was tested in the pentylenetetrazol (PTZ) and maximal electroshock (MES) models of epilepsy in mice. When administered concurrently, STP (100 mg/kg ip) significantly reduced the number of animals that exhibited PTZ-induced seizures and eliminated the extensor reflex of maximal electric-induced seizures test in 50% of the experimental animals. In addition, it showed protection in the PTZ test by diminishing the death rate.

Antianxiety and antidepressant effects of riparin III from Aniba riparia (Nees) Mez (Lauraceae) in mice.

This work presents behavioral effects of methyl ethers of N-(2,6-dihydroxybenzoyl) tyramine (riparin III) isolated from the unripe fruit of Aniba riparia on the open field, elevated plus maze (EPM), rotarod, hole board, barbiturate-induced sleeping time, tail suspension and forced swimming tests in mice. Riparin III was administered intraperitoneally to male mice at single doses of 25 and 50 mg/kg. The results showed that riparin III with both doses had no effects on spontaneous motor activity in mice or in the rotarod test, but decreased the number of grooming and rearing. At the dose of 50 mg/kg, riparin III increased the number of entries in the open arms of the EPM test as compared with control. Similarly, in the hole-board test, both doses increased the number of head dips. There was a reduction on the sleeping latency with both doses and a prolongation of the pentobarbital-induced sleeping time with the dose of 25 mg/kg. In the tail suspension test, similar to imipramine (30 mg/kg), riparin III at the dose of 50 mg/kg presented a reduction in the immobility time. In the forced swimming test, both doses of riparin III decreased the immobility time. These results showed that riparin III potentiated the barbiturate-induced sleeping time and presented antidepressant- and anxiolytic-like effects.

Studies on the mechanism of spasmolytic activity of (O-methyl-)-N-(2,6-dihydroxybenzoyl)tyramine, a constituent of Aniba riparia (Nees) Mez. (Lauraceae), in rat uterus, rabbit aorta and guinea-pig alveolar leucocytes.

The mechanism of action of a nonspecific smooth muscle relaxant, (O-methyl-)-N-(2,6-dihydroxybenzoyl)tyramine (riparin), a constituent of Aniba riparia (Nees) Mez. (Lauraceae) was studied in relation to Ca2+ metabolism in smooth muscle tissues and in guinea-pig alveolar leucocytes. In rat depolarized uterus, riparin inhibited in a reversible and noncompetitive manner CaCl2-induced contraction, a response mediated through voltage-dependent Ca2+ channels. The pD2 value (mean +/- s.e.m.) for riparin was 4.98 +/- 0.06. When compared with sodium nitroprusside (IC50 2.5 microM), an antagonist of receptor-operated Ca2+ channels, riparin was ineffective in suppressing noradrenaline-induced sustained contractions of rabbit aortic strips. However, in the aorta, the compound inhibited intracellular calcium-dependent transient contractions of noradrenaline and riparin (IC50 10.1 microM) was approximately two and a half times more potent than procaine (IC50 25.5 microM) a known inhibitor. In guinea-pig alveolar leucocytes, riparin (IC50 3.2 microM) inhibited intracellular Ca2+ accumulation induced by the calcium ionophore A23187. The results suggest that the inhibition of Ca2+ influx and of Ca2+ release from intracellular stores contribute to the spasmolytic effects of riparin, which may not involve cyclic AMP generation as the levels of this nucleotide were not increased in alveolar macrophages treated with riparin (10-100 microM).

Modification of Ca2+ metabolism in the rabbit aorta as a mechanism of spasmolytic action of warifteine, a bisbenzylisoquinoline alkaloid isolated from the leaves of Cissampelos sympodialis Eichl. (Menispermaceae).

The regulation of intracellular Ca2+ as a mechanism of spasmolytic activity of a bisbenzylisoquinoline alkaloid, warifteine, isolated from the leaves of Cissampelos sympodialis, Eichl (Menispermaceae) was studied in the rabbit aorta. Warifteine (pD2' 4.12 +/- 0.09) similar to verapamil (pD2' 6.89 +/- 0.05) antagonized, in a noncompetitive and reversible manner, KCl-induced contractions, mediated by Ca2+ entry through voltage-operated channels. Noradrenaline-induced sustained contractions mediated by Ca2+ entry through receptor-operated channels were also inhibited by warifteine (IC50 6.03 x 10(-5) M) and the standard agent sodium nitroprusside (IC50 1.9 x 10(-8) M). In Ca(2+)-free medium, the alkaloid reduced the intracellular Ca(2+)-dependent transient contraction to noradrenaline by inhibiting the release of Ca2+ (IC50 2.6 x 10(-5) M) from the stores and the refilling (IC50 1.9 x 10(-5) M) of the intracellular stores. The standard agent, procaine, also inhibited the release of Ca2+ (IC50 3.2 x 10(-5) M) but had no significant effect on Ca2+ uptake into the stores. Warifteine failed to affect intracellular Ca2+ stores sensitive to caffeine, while procaine inhibited (IC50 7.9 x 10(-4) M) the release of Ca2+ from these stores. The results indicate that warifteine may cause muscle relaxation by inhibiting Ca2+ channels and by modifying the intracellular Ca2+ stores sensitive to noradrenaline.

Calcium antagonism and the vasorelaxation of the rat aorta induced by rotundifolone.

The vasorelaxing activity of rotundifolone (ROT), a major constituent (63.5%) of the essential oil of Mentha x villosa, was tested in male Wistar rats (300-350 g). In isolated rat aortic rings, increasing ROT concentrations (0.3, 1, 10, 100, 300, and 500 microg/ml) inhibited the contractile effects of 1 microM phenylephrine and of 80 or 30 mM KCl (IC50 values, reported as means +/- SEM = 184 +/- 6, 185 +/- 3 and 188 +/- 19 microg/ml, N = 6, respectively). In aortic rings pre-contracted with 1 microM phenylephrine, the smooth muscle-relaxant activity of ROT was inhibited by removal of the vascular endothelium (IC50 value = 235 +/- 7 microg/ml, N = 6). Furthermore, ROT inhibited (pD2 = 6.04, N = 6) the CaCl2-induced contraction in depolarizing medium in a concentration-dependent manner. In Ca2+-free solution, ROT inhibited 1 microM phenylephrine-induced contraction in a concentration-dependent manner and did not modify the phasic contractile response evoked by caffeine (20 mM). In conclusion, in the present study we have shown that ROT produces an endothelium-independent vasorelaxing effect in the rat aorta. The results further indicated that in the rat aorta ROT is able to induce vasorelaxation, at least in part, by inhibiting both: a) voltage-dependent Ca2 channels, and b) intracellular Ca2+ release selectively due to inositol 1,4,5-triphosphate activation. Additional studies are required to elucidate the mechanisms underlying ROT-induced relaxation.

Pharmacological studies on Aristolochia papillaris Mast. (Aristolochiaceae).

The non-specific and reversible smooth muscle relaxant activities of the ethanolic extract (EE) of Aristolochia papillaris Mast., a fraction of EE containing tertiary alkaloids (TAF) and of 3 compounds isolated from TAF are reported. In the non-pregnant rat uterus, EE and TAF inhibited both the oxytocin-induced contractions and the amplitude of rhythmic spontaneous contractions. The IC50 values for EE and TAF were 0.91 and 0.22 microgram/ml, respectively in the first experiments while the corresponding values were 1.0 and 0.17 microgram/ml in the second case. The rhythmic contractions of the uterus obtained from 21-day pregnant rats were also reduced by EE and TAF with IC50 values of 25.5 and 11.2 micrograms/ml, respectively. The relaxation of isolated guinea pig trachea produced by EE and TAF were also observed with the compounds moupinamide, coclaurine and isoboldine isolated from TAF. The IC50 values of these compounds were 1.58 x 10(-4) M, 3.98 x 10(-4) M and 7.10 x 10(-4) M, respectively. Propranolol significantly antagonized the effects of coclaurine and isoboldine but failed to inhibit the responses to moupinamide which suggests that the plant constituents produce muscle relaxation by beta-adrenoceptor-dependent and -independent mechanisms.

Central depressant effects of reticuline extracted from Ocotea duckei in rats and mice.

Neuropharmacological studies were carried out with reticuline, a benzylisoquinoline alkaloid, isolated from Ocotea duckei Vattimo. It was found that reticuline (50-100 mg/kg i.p.) produced alteration of behaviour pattern, prolongation of pentobarbital-induced sleep, reduction in motor coordination and D-amphetamine-induced hypermotility and suppression of the conditioned avoidance response. These observations suggest that reticuline possesses potent central nervous system depressant action.

Anti-inflammatory actions of tannins isolated from the bark of Anacardium occidentale L.

A mixture of tannins (hydrolysable and non-hydrolysable) obtained from the bark of Anacardium occidentale L., on i.p. injection, demonstrated apparent anti-inflammatory activity in carrageenan- and dextran-induced rat paw oedemas, cotton pellet granuloma test and adjuvant-induced polyarthritis in rats. At higher doses orally administered tannins also had activity in carrageenan paw oedema and adjuvant arthritis experiments. The tannins i.p. also inhibited acetic acid-induced "writhing responses" in mice and were found to antagonise the permeability-increasing effects in rats of certain mediators of inflammation and to inhibit the migration of leucocytes to an inflammatory site. While not appearing to act by the release of adrenal hormones, tannins may produce effects in a non-specific manner by their astringent properties on cell membranes thus affecting cell functions. The above results should be considered while studying the anti-inflammatory actions of plant extracts which contain tannins.

Probable mechanism of hypoglycemic activity of bassic acid, a natural product isolated from Bumelia sartorum.

Bassic acid, an unsaturated triterpene acid isolated from an ethanol extract of Bumelia sartorum rootbark, elicited significant hypoglycemic activity in alloxan-diabetic rats and altered the pattern of glucose tolerance in these animals. In addition, bassic acid treatment increased significantly the glucose uptake process and glycogen synthesis in isolated rat diaphragm. Bassic acid treatment increased plasma insulin levels significantly in alloxan-diabetic rats. It is suggested that the hypoglycemia activity of bassic acid may be mediated through enhanced secretion of insulin from the pancreatic beta-cells.

Antimicrobial activity of benzylisoquinoline alkaloids from Annona salzmanii D.C.

Bark of Annona salzmanii D.C. (Annonaceae), used in Brazilian folk medicine, was found to contain four benzylisoquinoline alkaloids, namely reticuline, anonaine, laurelliptine and isoboldine. Only anonaine possesses some antibacterial property while all four alkaloids show some antifungal activity.

CNS pharmacological effects of the total alkaloidal fraction from Albizia inopinata leaves.

The total alkaloidal fraction of Albizia inopinata leaves (FLA) was investigated for its central nervous system (CNS) effects. FLA (10 mg/kg, i.p.) significantly reduced (45%) the locomotor activity in mice. In addition, it inhibited the conditioned avoidance response behavior and induced ptosis in rats. On the other hand, FLA did not exert significant effect on catalepsy, but potentiated the haloperidol-induced catalepsy. No effect was observed on sleep induced by sodium pentobarbital or apomorphine-induced stereotypes.