Assessment of anxiolytic and panicolytic effects of dichloromethane fraction from stems of Kielmeyera coriacea.

Kielmeyera coriacea Mart. (Calophyllaceae) is known popularly as "Pau Santo". The hydroethanolic extract (HE) of Kielmeyera coriacea stems and its semi-pure dichloromethane (DCM) constituent produced an antidepressant-like effect in rats. The purpose of this study was to investigate the effects of repeated administration (21 days) by gavage of the DCM fraction (5, 10 or 15mg/kg) in rats submitted to the elevated T-maze (ETM), a model of generalized anxiety and panic disorders. The tricyclic antidepressant imipramine (15mg/kg) was used as a positive control. Rat locomotion was assessed using the open field test (OFT) following each drug treatment. The 2-hydroxy-1-methoxyxanthone (1), aucuparin (2), swertinin (3), 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (4) and 1,3,5-trihydroxy-2-(3-methylbut-2-enyl)-xanthone (5) were identified in DCM fraction, and suggest that the xanthone (4) is related with the antidepressant-like profile of this plant. Pharmacological evaluation showed that DCM fraction (10 and 15 mg/kg) decreased the inhibitory avoidance latency from the closed arm and increased the one-way escape latency from the open arm in the ETM, which is indicative of anxiolytic and panicolytic effects, respectively, as occurs with the positive control, imipramine (15 mg/kg), when compared to their control group (vehicle). Locomotor activity was not significantly altered by the different treatments. This study suggests that the DCM fraction from stems of Kielmeyera coriacea can be an important therapeutic alternative in the treatment of anxiety disorders, such as generalized anxiety and panic disorders.

Acute and subchronic toxicological evaluation of the semipurified extract of seeds of guaraná (Paullinia cupana) in rodents.

We evaluated the toxicity of a semipurified extract (EPA fraction, containing caffeine and several flavan-3-ols and proanthocyanidins) of seeds of the native Amazon plant Paullinia cupana (guaraná) in rodents. Acute toxicity was tested in male Swiss mice, which received different doses orally (OR) and intraperitoneally (ip); control groups received water. These tests produced acute mortality, with LD(50) of 1.825 g/kg (OR) and 0.827 g/kg (ip), and a significant weight decrease in lungs of mice receiving a dose of 0.1g/kg. In the repeated-dose toxicity test, the EPA was administered OR daily to male and female Wistar rats at doses of 30, 150, and 300 mg/kg/day/90 days. Their behavior, mortality, weight changes, laboratory tests, and the weights and histopathology of organs were evaluated. No rats died during the tests. Males dosed at 150 or 300 mg/kg gained weight more slowly and lost kidney weight (absolute and relative weights, compared to the control group). Hematological and biochemical tests showed few changes, differing somewhat between males and females; the histopathological evaluation indicated no significant changes. These results indicate that the EPA fraction of guaraná caused no toxicity in rats at the smallest dose evaluated (30 mg/kg). No other species was evaluated.

Effect of xanthone from Kielmeyera coriacea stems on serotonergic neurons of the median raphe nucleus.

Kielmeyera coriacea Mart. (Clusiaceae), known as "Pau Santo", is used to treat several tropical diseases. The hydroethanolic extract (HE) of Kielmeyera coriacea stems and its semi-pure dichloromethane constituent (DCM) produced an anti-immobility effect in rats submitted to the forced swimming test (FST), suggesting a antidepressant-like profile. This study evaluated the effect of intra-median raphe nucleus (MRN) microinjection of 1,3,7-trihydroxy-2-(3-methylbut-2-enyl)-xanthone, present in large quantity in the HE from Kielmeyera coriacea stems, on immobility behaviour in the FST in rats. The effects of xanthone were compared with intra-MRN microinjections of Way100635 (5-HT1A antagonist) or (+) 8-OH-DPAT (5-HT1A agonist). Locomotor activity in the open-field test (OFT) was evaluated as a complementary measure. Xanthone (0.3ng) or Way100635 (2.5microg) reduced, whereas (+) 8-OH-DPAT (5.0microg) increased immobility time in the FST. Way100635 (2.5 or 5.0microg) completely reversed the effects of (+) 8-OHDPAT (5.0microg), and potentiated the anti-immobility effect of the ineffective dose of xanthone (0.2ng) in the FST. The association of effective doses of (+) 8-OH-DPAT (5.0microg) and xanthone (0.3ng) annulled the effect of each compound on immobility time. These results suggest that xanthone acts as an antagonist at 5-HT1A autoreceptors in MRN and increases serotonin (5-HT) availability in projection regions, proving to be a prototype drug that may be useful in mood isorders such as depression, or indeed be a beneficial adjunctive treatment improving the efficacy and/or accelerating the effects of antidepressant drugs in patients with major depression.

Effect of lyophilized extracts from guaraná seeds [Paullinia cupana var. sorbilis (Mart.) Ducke] on behavioral profiles in rats.

The aim of the present study was to investigate the pharmacological properties of the crude lyophilized extract (EBPC) of Paullinia cupana seeds (guaraná) and the semi-purified extracts (EPA and EPB) after acute or chronic administration by the oral route in rats. Anxiolytic-like, antidepressant-like and motor stimulant effects were evaluated using the plus maze (PMT), forced swimming (FST) and open field (OFT) tests, respectively. Acute or chronic administration of EBPC (3.0, 30.0 or 60.0 mg/kg) did not alter the percentage of entries or the time spent in the open arm in the PMT. In the FST, chronic treatment with 30.0 mg EBPC/kg and 4.0 mg EPA/kg extract decreased the immobility time similarly to the antidepressant reference drug, imipramine (20.0 mg/kg). Locomotor activity in the OFT was not increased by these extracts. Caffeine (10.0 mg/kg) significantly reduced the immobility time in the FST, but increased locomotor activity in the OFT, indicating psychostimulant activity. The EPB extract did not induce any effect after acute or chronic treatment in the different models used. The present results suggest that the crude EBPC extract and EPA extract produced an antidepressant-like effect after long-term administration.

Avaliação físico-química de sementes de guaraná secas por diferentes métodos / Physico-chemical evaluation of guarana seeds dried by different methods

Amostras de sementes de guaraná provenientes da região amazônica e secas por diferentes métodos, foram analisadas por técnicas farmacopéicas e outras, e o extrato liofilizado foi analisado por cromatografia líquida de alta eficiência (CLAE). Realizou-se o doseamento de metilxantinas (MX) e dos taninos totais (TT), através de métodos espectrofotométricos, para a comparação das amostras. O maior teor de MX foi obtido com as sementes secas em tacho metálico por 4 h com adição de água, enquanto que o maior teor de TI foi obtido com a amostra torrada em tacho metálico por 4 h sem água. A análise cromatográfica por CLAE apresentou tempos de retenção para catequina, epicatequina e cafeína de 6,17; 8,85 e 11,91 min, respectivamente.

Contrai quality of the vegetable drug "guaraná", with samples of seeds obtained from Amazonian area, were carried out by pharmacopoeial assays, and the extract were analysed by HPLC. The determination of methylxantines (MX) and total tannins (TI) was performed by spectrophotometric methods. The most yield of MX were showed by the sample GTIB and for TI the best result were obtained by the sample GTIM4s. The analysis by HPLC showed retention times for catechin, epicatechin and cafteine of 6.17,8.85 and 11.91 min, respectively.

Preliminary evaluation of Kielmeyera coriacea leaves extract on the central nervous system.

The hydroalcoholic extract of Kielmeyera coriacea leaves was evaluated for effects on the central nervous system by means of behavioral models which included forced swimming, open-field and elevated plus-maze tests. According to the results, K. coriacea leaves extract is effective as anxiolytic but not as an antidepressant drug.

Gastric antiulcerogenic effects of Stryphnodendron adstringens in rats.

The antiulcer activity of the total extract and the fractions of Stryphnodendron adstringens was studied in rats and compared with that of cimetidine. Ulcers were induced in rats by means of three experimental models: acute stress, acidified-ethanol and indomethacin. The total extract and the fractions were found to have significant antiulcer activity in the case of the acute stress and acidified-ethanol models. These findings support the use of S. adstringens extracts in the treatment of gastric lesions.

Role of the amygdala and periaqueductal gray in anxiety and panic.

The amygdala (AM) and the periaqueductal gray (PAG) represent the rostral and the caudal pole, respectively, of a longitudinally organized neural system, that is responsible for the integration of behavioral and physiological manifestations of defensive reactions against innate and learned threats. Microinjection of benzodiazepine (BZD) anxiolytics, GABAA receptor agonists or 5-HT receptor antagonists into the AM has anxiolytic effects in conflict tests and other models of conditioned fear, while similar administration of 5-HT or of a 5-HT1A receptor agonist has anxiogenic effects. On the other hand, in the test of electrical stimulation of the PAG, microinjection of 5-HT, 5-HT mimetics, or of drugs that enhance the action of endogenous 5-HT into the same brain area has an antiaversive effect, like BZD and GABAA agonists. Furthermore, microinjection of midazolam, of the NMDA receptor antagonist AP-7, or of the 5-HT1A/1B receptor blocker propranolol increased the exploration of the open arms of the elevated plus-maze, having therefore an anxiolytic effect. These results point to an inhibitory role of the GABA-BZD system in both the AM and the PAG. In contrast, 5-HT seemingly enhances conditioned fear in the AM, while inhibiting unconditioned fear in the PAG. Thus, 5-HT2/1C antagonists reportedly release punished behavior when injected into the AM, whereas they antagonized the antiaversive effect of 5-HT, zimelidine and 5-HT1A/1B receptor blockers in the PAG. Since reported clinical studies revealed that one of such compounds, ritanserin, relieves generalized anxiety but tends to aggravate panic disorder, a relationship may be established between the AM and anxiety and the PAG and panic.

Microinjection of propranolol into the dorsal periaqueductal gray causes an anxiolytic effect in the elevated plus-maze antagonized by ritanserin.

The 5-HT1A/1B receptor antagonist propranolol was injected into the dorsal periaqueductal gray (DPAG) of rats exposed to the elevated plus-maze in order to investigate the participation in anxiety of 5-HT mechanisms operating in this brain region. Microinjection of D,L- or L-propranolol into the DPAG increased the percentage of total arm entries without affecting the total number of entries into either open or enclosed arms of the maze, an effect characteristic of anxiolytic drugs injected systemically. The doses of 5 nmol L-propranolol and 10 nmol D,L-propranolol caused anxiolytic effects of comparable magnitude, while the doses of 2.5 nmol of the former and 5 nmol of the latter were ineffective. Therefore, the L-isomer is likely to be the main one responsible for the pharmacological activity observed. In addition, the anxiolytic effect of 10 nmol D,L-propranolol was antagonized by 10 nmol of the 5-HT2/1C receptor antagonist ritanserin, previously injected into the DPAG. The present as well as previously reported results suggest that the anxiolytic effect of propranolol injected into the DPAG is due to increased release of 5-HT acting on post-synaptic 5-HT2 receptors, resultant from blockade of 5-HT1B autoreceptors that inhibit amine release from serotonergic nerve endings.

Stereoselectivity of the anxiolytic effect of propranolol microinjected into the dorsal midbrain central gray.

In a previous study we have shown that microinjection of d,1-propranolol into the dorsal midbrain central gray of the rat causes an anxiolytic effect in the elevated plus-maze model which is likely to be mediated by endogenous 5-hydroxytryptamine. In the present experiment, the effects of 1- and d,1-propranolol were compared under the same experimental conditions. Both the 1-isomer and the racemic mixture increased the percentage of open arm entries without affecting the total number of entries into either open or enclosed arms of the maze, thus reproducing the selective anxiolytic effect previously described. The doses of 5 nmol 1-propranolol and 10 nmol d,1-propranolol caused anxiolytic effects of comparable magnitude, while the doses of 2.5 nmol of the former and 5 nmol of the latter were ineffective. Therefore, the 1-isomer was nearly twice as potent as the racemic mixture, thus being responsible for the pharmacological activity observed. These results are compatible with the proposal that propranol blocks stereospecific autoreceptors in serotonergic nerve endings that inhibit neurotransmitter release.

Behavioral effects of 5-HT receptor ligands in the aversive brain stimulation, elevated plus-maze and learned helplessness tests.

In order to illustrate the use of animal models in the study of the anxiolytic and antidepressant properties of drugs acting on 5-HT receptors, a series of experiments is described. With electrical stimulation of the midbrain central gray (CG), an aversive area of the brain, the 5-HT-1 receptor antagonist propranolol raised the aversive threshold in a dose-dependent way, following its microinjection into the CG. This antiaversive effect of propranolol, which is similar to that of benzodiazepine anxiolytics, was prevented by microinjection into the same brain site of the 5-HT-2 receptor blocker ritanserin. Ritanserin itself and the 5-HT-1A receptor ligand ipsapirone caused either little or no effect. In another animal model of anxiety, the elevated plus-maze, intra-CG propranolol also caused an anxiolytic-like effect, antagonized by ritanserin, indicating a 5-HT mediation. However, systemically injected isamoltane, a congener of propranolol, was ineffective in the elevated plus-maze, whereas ipsapirone caused an anxiolytic effect. Ritanserin was again inactive. Finally, both ipsapirone as well as another 5-HT-1A receptor ligand BAY R 1531, given IP, reversed the learning deficit resulting from exposure to uncontrollable foot-shocks, an effect characteristic of antidepressant drugs.

Stereoselectivity of the anxiolytic effect of propranolol microinjected into the dorsal midbrain central gray

In a previous study we have shown that microinjection of d,I-propranolol into the dorsal midbrain central gray of the rat causes an anxiolytic effect in the elevated plus-maze model which is lilkely to be mediated by endogenous 5-hydroxytryptamine. In the present experiment, the effects of 1- and d,1-propranolol were compared under the same experimental conditions. Both the I-isomer and the racemic mixture increased the percentage of open arm entries without affecting the total number of entries into either open or enclosed arms of the maze, thus reproducing the selective anxiolytic effect previously described. The doses of 5 nmol 1-propanolol and 10 nmol d,1-propranolol caused anxiolytic effects of comparable magnitude, while the doses of 2,5 nmol of the former and 5 nmol of the latter were ineffective. Therefore, the 1-isomer was nearly twice as potent as the racemic mixture, thus being responsible for the pharmacological activity observed. These results are compatible with the proposal that propranol blocks stereospecific autoreceptors in serotonergic nerve endings that inhibit neurotransmitter release

Serotonergic mediation of the anxiolytic effect of intracerebrally injected propranolol measured in the elevated plus-made

the effect of intracerebrally injected propranolol was measured in the elevated plus-maze, an animal model of anxiety. Microinjection of 10 nmol of propranolol into the dorsal midbrain central gray of the perventage of open arm entries, without affecting the total number of arm entries.This selective anxiolytic effect of propranolol was antagonized by 10 nmol of ritanserin, also injected into the dorsal midbrain. The same dose of ritanserin, given alone, did not affect the percentage of open arn entries, thought it tended to decrease the total number of entries, an indication of unspecific behavioral depression. Since propranolol is a sterospecific antagonist of presynaptic serotpnin (5-HT) antoreceptors and ritanserin is a selective blocker of type 2 5-HT recptors, the present results suggest that the anxiolytic action of propranolol in the midbrain central gray is due to release of endogenous 5-HT acting upon 5-HT2 receptors

Serotonergic mediation of the anxiolytic effect of intracerebrally injected propranolol measured in the elevated plus-maze.

The effect of intracerebrally injected propranolol was measured in the elevated plus-maze, an animal model of anxiety. Microinjection of 10 nmol of propranolol into the dorsal midbrain central gray of the rat increased the percentage of open arm entries, without affecting the total number of arm entries. This selective anxiolytic effect of propranolol was antagonized by 10 nmol of ritanserin, also injected into the dorsal midbrain. The same dose of ritanserin, given alone, did not affect the percentage of open arm entries, though it tended to decrease the total number of entries, an indication of unspecific behavioral depression. Since propranolol is a stereospecific antagonist of presynaptic serotonin (5-HT) autoreceptors and ritanserin is a selective blocker of type 2 5-HT receptors, the present results suggest that the anxiolytic action of propranolol in the midbrain central gray is due to release of endogenous 5-HT acting upon 5-HT2 receptors.

Mediation by serotonin of the antiaversive effect of zimelidine and propranolol injected into the dorsal midbrain central grey.

Previously reported results indicate that serotonin (5-HT) inhibits the neural sub strate of aversion in the dorsal midbrain central grey (DCG) of the rat. In addition, the present results show that microinjection of the 5-HT uptake inhibitor zimelidine (100 nmol) into the DCG of rats with chronically implanted chemitrodes raised the threshold of aversive electrical stimulation. This antiaversive effect of zimelidine was antagonized by pretreatment with the 5-HT(2) receptor blocker ritanserin (10 nmol), also microinjected into the DCG. In contrast, the antiaversive effect of the benzodiazepine agonist midazolam (40 nmol) was unaffected by ritanserin. Propranolol (2.2, 4.4 and 8.8 nmol) raised the aversive threshold in a dose-depen dent way following its injection into the DCG. The antiaversive effect of 4.4 nmol of propranolol was antagonized by previous administration of ritanserin (10 nmol). Moreover, combined administration of zimelidine (100 nmol) followed by propranolol (4.4 nmol) caused an anti aversive effect which was equivalent to the sum of the effect of each drug alone. These results indicate that the antiaversive effect of intracerebrally injected zimelidine and propranolol is mediated by endogenous 5-HT, through activation of 5-HT(2) receptors.

GABAA receptors in the midbrain central grey mediate the antiaversive action of GABA.

Intracerebral injection of the GABAA agonists muscimol (1 nmol), isoguvacine (1 nmol) or THIP (1, 2 and 4 nmol) in rats with chemitrodes implanted in the dorsal midbrain central grey raised the threshold electrical current for inducing escape behaviour. The effect of THIP was dose-dependent. In contrast, the GABAB agonist baclofen (10 and 100 nmol) did not affect the aversive threshold. Furthermore, pretreatment with baclofen (10 nmol and 100 nmol) did not significantly change the effect of THIP (2 nmol). These results indicate that the antiaversive action of GABA in the midbrain central grey is mediated by GABAA but not by GABAB receptors.

Modulation of the brain aversive system by GABAergic and serotonergic mechanisms.

Experiments performed in our laboratory, using electrical stimulation combined with microinjection of drugs in the dorsal midbrain central grey (CG) of the rat, evidenced that direct stimulation of GABA receptors with locally administered gamma-aminobutyric acid (GABA) or the GABAA receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, isoguvacine and muscimol raised the aversive threshold, defined as the lowest electrical current intensity inducing flight or escape behaviour when applied to the dorsal CG. The GABAB receptor agonist baclofen was ineffective. Also, enhancement of endogenous GABA action through local injection of the benzodiazepines chlordiazepoxide and midazolam or of pentobarbital resulted in anti-aversive effects. Ro 15-1788 antagonized both chlordiazepoxide and midazolam, suggesting benzodiazepine receptor mediation. In contrast to pro-GABAergic drugs, microinjection of the GABA antagonists bicuculline and picrotoxin into the CG elicited flight behaviour, like the electrical stimulation. Similar experiments with drugs influencing serotonergic neurotransmission evidenced that intra-CG microinjection of serotonin (5-HT) or of the direct 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine increased the aversive threshold. The anti-aversive effect of 5-HT was potentiated by the selective inhibitor of 5-HT neuronal uptake, zimelidine. Also, the latter drug increased the aversive threshold when given alone. The anti-aversive effect of 5-HT was antagonized by local pretreatment with either metergoline or ketanserin, the latter being a selective blocker of 5-HT2 receptors. In contrast to the GABA antagonists mentioned above, the 5-HT receptor blockers did not evoke aversive behaviour per se. Therefore, both GABAergic and serotonergic mechanisms are likely to play an inhibitory role in the dorsal CG integrating aversive behaviour. The former seem to act tonically, whereas 5-HT would act in a phasic way. The implications of these results for the pathophysiology and drug treatment of chronic anxiety, panic states and pain disorders are briefly discussed.

Modulation of the brain aversive system by GABAergic and serotonergic mechanisms.

Experiments performed in our laboratory, using electrical stimulation combined with microinjection of drugs in the dorsal midbrain central grey (CG) of the rat, evidenced that direct stimulation of GABA receptors with locally administered gamma-aminobutyric acid (GABA) or the GABAA receptor agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol, isoguvacine and muscimol raised the aversive threshold, defined as the lowest electrical current intensity inducing flight or escape behaviour when applied to the dorsal CG. The GABAB receptor agonist baclofen was ineffective. Also, enhancement of endogenous GABA action through local injection of the benzodiazepines chlordiazepoxide and midazolam or of pentobarbital resulted in anti-aversive effects. Ro 15-1788 antagonized both chlordiazepoxide and midazolam, suggesting benzodiazepine receptor mediation. In contrast to pro-GABAergic drugs, microinjection of the GABA antagonists bicuculline and picrotoxin into the CG elicited flight behaviour, like the electrical stimulation. Similar experiments with drugs influencing serotonergic neurotransmission evidenced that intra-CG microinjection of serotonin (5-HT) or of the direct 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine increased the aversive threshold. The anti-aversive effect of 5-HT was potentiated by the selective inhibitor of 5-HT neuronal uptake, zimelidine. Also, the latter drug increased the aversive threshold when given alone. The anti-aversive effect of 5-HT was antagonized by local pretreatment with either metergoline or ketanserin, the latter being a selective blocker of 5-HT2 receptors. In contrast to the GABA antagonists mentioned above, the 5-HT receptor blockers did not evoke aversive behaviour per se. Therefore, both GABAergic and serotonergic mechanisms are likely to play an inhibitory role in the dorsal CG integrating aversive behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of GABA in the anti-aversive action of anxiolytics.

The above results with intracerebral drug injection and electrical brain stimulation in rats indicate that enhancement of GABAergic activity in the dorsal CG or in the MH raises the threshold of electrical stimulation inducing aversive behavior when applied to these brain areas. Conversely, drug-induced reduction of GABA action in the dorsal CG or in the MH leads to aversive-like behavioral changes. Therefore, GABAergic fibers seem to exert tonic inhibition on neuronal groups in the CG and MH integrating aversive behavioral states. Anxiolytics may cause anti-aversive effects by enhancing this GABAergic modulation. As discussed elsewhere, serotonergic and opioid mechanisms are also likely to operate in periventricular brain areas. In conjunction with GABAergic mechanisms, they may be involved in the pathophysiology of anxiety, panic attacks and pain, as well as in the therapeutic action of anxiolytics, anti-panic drugs and centrally-acting analgesics.

Benzodiazepine receptors in the periaqueductal grey mediate anti-aversive drug action.

The microinjection of 80, 160 and 320 nmol chlordiazepoxide (CDP) as well as of 20, 40 and 80 nmol midazolam (MDZ) into the dorsal midbrain of rats bearing chronically implanted chemitrodes raised the threshold electrical current inducing escape behaviour by stimulating the dorsal periaqueductal grey matter (DPAG). Parallel linear regressions were obtained by plotting the log dose against drug-induced increases in escape threshold, MDZ being 3.55 times more potent than CDP (95% confidence limits 1.21 and 8.57). Local pretreatment with 80 nmol of the benzodiazepine antagonist Ro 15-1788 blocked the anti-aversive effect of either 160 nmol CDP or 40 nmol MDZ. The same dose of Ro 15-1788 was ineffective when given alone. These results suggest that the anti-aversive action of CDP and MDZ is due to their combination with benzodiazepine receptors in the DPAG.