Anxiolytic activity and active principles of Piper amalago (Piperaceae), a medicinal plant used by the Q'eqchi' Maya to treat susto, a culture-bound illness.

ETHNOPHARMACOLOGICAL RELEVANCE: The medicinal plant, Piper amalago L. (Piperaceae), is used traditionally by Q'eqchi' Maya healers for the treatment of "susto" a culture-bound syndrome. Previous research suggests that susto symptoms may be a manifestation of anxiety. The objectives were to characterize the effect of ethanolic extract of P. amalago in behavioral assays of anxiety at doses representative of traditional use and to isolate active principles. MATERIALS AND METHODS: Rats treated orally with low dose ethanolic extracts of P. amalago leaves (8-75mg/kg) were tested in several behavioral paradigms including the elevated plus maze (EPM), social interaction (SI), and conditioned emotional response (CER) tests, and compared to diazepam, a positive control. The active anxiolytic principle was isolated by bioassay guided isolation using an in vitro GABAA competitive binding assay. RESULTS: Extracts had significant anxiolytic activity in all behavioral tests, with the strongest activity in the SI and the CER paradigms. In an in vitro GABAA competitive binding assay, a 66.5µg/mL concentration of P. amalago ethanol extract displaced 50% of the GABAA-BZD receptor ligand [(3)H]-Flunitrazepam. Bioassay-guided fractionation identified a furofuran lignan, a molecule with structural similarity to yangambin, with high affinity for the GABAA-BZD receptor as the principle bioactive. CONCLUSION: The results suggest that the ethnobotanical use of this plant may have a pharmacological basis in its anxiolytic activity, as demonstrated in animal behaviour tests.

Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system.

In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed.

Anxiety responses, plasma corticosterone and central monoamine variations elicited by stressors in reactive and nonreactive mice and their reciprocal F1 hybrids.

Stressor-provoked anxiety, plasma corticosterone, and variations of brain monoamine turnover are influenced by genetic factors, but may also be moderated by early life experiences. To evaluate the contribution of maternal influences, behavioral and neurochemical stress responses were assessed in strains of mice that were either stressor-reactive or -resilient (BALB/cByJ and C57BL/6ByJ, respectively) as well as in their reciprocal F(1) hybrids. BALB/cByJ mice demonstrated poorer maternal behaviors than did C57BL/6ByJ dams, irrespective of the pups being raised (inbred or F(1) hybrids). The BALB/cByJ mice appeared more anxious than C57BL/6ByJ mice, exhibiting greater reluctance to step-down from a platform and a greater startle response. Although the F(1) behavior generally resembled that of the C57BL/6ByJ parent strain, in the step-down test the influence of maternal factors were initially evident among the F(1) mice (particularly males) with a BALB/cByJ dam. However, over trials the C57BL/6ByJ-like behavior came to predominate. BALB/cByJ mice also exhibited greater plasma corticosterone elevations, 5-HT utilization in the central amygdala (CeA), and greater NE turnover in the paraventricular nucleus of the hypothalamus (PVN). Interestingly, among the F(1)'s corticosterone and 5-HIAA in the CeA resembled that of the BALB/cByJ parent strain, whereas MHPG accumulation in the PVN was more like that of C57BL/6ByJ mice. It seems that, to some extent, maternal factors influenced anxiety responses in the hybrids, but did not influence the corticosterone or the monoamine variations. The inheritance profiles suggest that anxiety was unrelated to either the corticosterone or monoamine changes.

Evaluation of anxiolytic properties of Gotukola--(Centella asiatica) extracts and asiaticoside in rat behavioral models.

The ayurvedic medicinal plant Gotukola (Centella asiatica) was evaluated for its anxiolytic properties. Specifically, this study assessed the effects of: Gotukola plant materials of different genotypic origin; hexane, ethyl acetate and methanol extracts of Gotukola; and asiaticoside, a triterpenic compound isolated from Gotukola. Various paradigms were used to assess the anxiolytic activity, including the elevated plus maze (EPM), open field, social interaction, locomotor activity, punished drinking (Vogel) and novel cage tests. The EPM test revealed that Gotukola, its methanol and ethyl acetate extracts as well as the pure asiaticoside, imparted anxiolytic activity. Furthermore, the asiaticoside did not affect locomotor activity, suggesting these compounds do not have sedative effects in rodents.

Phytochemical and biological analysis of skullcap (Scutellaria lateriflora L.): a medicinal plant with anxiolytic properties.

The phytochemistry and biological activity of Scutellaria lateriflora L. (American skullcap) which has been traditionally used as a sedative and to treat various nervous disorders such as anxiety was studied. In vivo animal behaviour trials were performed to test anxiolytic effects in rats orally administered S. laterifolia extracts. Significant increases in the number of entries into the center of an "open-field arena"; number of unprotected head dips, number of entries and the length of time spent on the open arms of the Elevated Plus-Maze were found. The identification and quantification of the flavonoid, baicalin in a 50% EtOH extract (40 mg/g) and its aglycone baicalein in a 95% EtOH extract (33 mg/g), as well as the amino acids GABA in H2O and EtOH extracts (approximately 1.6 mg/g) and glutamine in a H2O extract (31 mg/g), was performed using HPLC. These compounds may play a role in anxiolytic activity since baicalin and baicalein are known to bind to the benzodiazepine site of the GABAA receptor and since GABA is the main inhibitory neurotransmitter.

Acoustic startle and fear-potentiated startle in rats selectively bred for fast and slow kindling rates: relation to monoamine activity.

The acoustic startle response, prepulse inhibition, fear-potentiated startle and monoamine activity induced by either, a novel stimulus or a cue previously paired with foot-shock (fear-conditioning), were assessed in rats selectively bred for differences in amygdala excitability (Fast vs. Slow kindling epileptogenesis). Comorbid differences of anxiety, which were dependent both on the rats' behavioural style and the kind of stressor, also characterized these strains. In the present investigation, Slow rats exhibited a greater startle reflex to noise relative to Fast rats, suggesting differences in generalized anxiety, but similar rates of startle habituation and prepulse inhibition. The fear-potentiated startle, however, was greater in Fast rats. When movement of the rat was restricted in a new environment, presentation of a novel stimulus (light) increased norepinephrine, dopamine and/or serotonin activity in brain regions typically associated with stressors (e.g. locus coeruleus, paraventricular hypothalamic nucleus). Generally, these effects were more pronounced in Fast rats, and norepinephrine utilization in the central amygdala was particularly highlighted in response to a conditioned fear stimulus. Thus, while generalized anxiety appeared greater in Slow rats, behavioural and central neurochemical reactivity in response to novel stimuli and to fear-eliciting stimuli, was greater in Fast rats. Similarly, basal dopamine activity in the prefrontal cortex was greater in Fast rats, but dopamine utilization elicited by a novel stimulus was more pronounced in Slow rats. This suggested that relative to Slow rats, dopamine neurons in prefrontal cortex of Fast rats do not react normally to environmental stimuli, and this phenomenon could lead to disturbances of attention or impulsivity.

Influence of acute tryptophan depletion on mood and immune measures in healthy males.

Depressive illness has been associated with variations of several aspects of immune functioning, as well as alterations of cytokine production in stimulated lymphocytes. In the present investigation we sought to determine whether pharmacologically-induced reductions of mood in healthy, male subjects would be associated with alterations in the levels of circulating IL-1 beta or IL-6 or to in vitro lymphocyte proliferation in response to T cell mitogens, PHA and Con A. Lowering tryptophan levels by means of a tryptophan-deficient amino acid mixture, which reduced plasma tryptophan and serotonin (5-HT) levels, produced a lowering of mood in a subset of male subjects (that had no personal or family history of depression) relative to subjects that received a balanced amino acid mixture. Correlational analyses revealed that the change of mood (particularly depression and anger) in subjects that received the tryptophan-free mixture was related to the extent of the tryptophan or 5-HT reductions. However, while fenfluramine administration resulted in recovery of tryptophan and 5-HT levels, this was not accompanied by recovery of mood. Furthermore, it was observed that the lowering of tryptophan levels and the reduced mood were not accompanied by variations of the cytokine levels or cell proliferation. Evidently, transient and modest alterations of 5-HT or mood induced by a tryptophan-free amino acid mixture were insufficient to promote variations of immune activity or circulating IL-1 beta or IL-6 levels. Even if depression were related to immune disturbances, the mood and 5-HT alterations associated with this type of manipulation may be too brief to promote immune changes comparable with those ordinarily associated with severe or chronic depressive illness.

Circulating lymphocyte subsets in major depression and dysthymia with typical or atypical features.

OBJECTIVE: Inconsistent results have been reported concerning circulating lymphocyte subsets in depression. To establish whether the immune alterations in depression could be related to neurovegetative symptoms, lymphocyte subsets were assessed in major depressive and dysthymic patients who exhibited either typical or atypical features (ie, the latter characterized by mood reactivity and reversed neurovegetative features). METHOD: Blood was collected from major depressive, atypical depressive, typical dysthymic, or atypical dysthymic patients and from nondepressed control subjects. Circulating lymphocyte subsets (CD3, CD4, CD8, CD19, CD16/CD56) were determined by flow cytometry. In a subset of patients, lymphocyte subsets were also determined after a 12-week course of antidepressant medication. RESULTS: Although T and B cell populations did not differ between the depressive subtypes and control subjects, circulating natural killer (NK) cells were elevated in depressive illness, and varied as a function of depressive subtype and sex. Among male patients, NK cells were elevated to a greater extent in typical than in atypical depression, and more so in major depressive than in dysthymic patients. Among female patients, circulating NK cells were lower than in male patients, and only among the typical major depressive patients did NK cells exceed those of controls. Normalization of NK cells occurred with successful pharmacotherapy. CONCLUSIONS: Depression may be associated with elevated levels of circulating NK cells. Although the neurovegetative features associated with depression, particularly altered eating, may have contributed to the elevated NK cells, depressive affect itself also contributed in this respect. However, the relative contributions of these factors varied between male and female patients.

Regulation of ingestion by CRF and bombesin-like peptides: distinct meal-related peptide level changes.

Bombesin (BN) and corticotropin-releasing factor (CRF) have both been shown to induce satiety in rats when injected centrally. The present study assessed temporal changes in the utilization of BN- and CRF-like peptides in relationship to feeding status, fluctuations that may indicate the physiological participation of these peptides in the regulation of feeding. Alterations in the endogenous levels of CRF- and BN-like peptides associated with the initial spontaneous meal of the nocturnal cycle were determined in 15 hypothalamic and extrahypothalamic brain nuclei in the following three groups of rats: 1) a preprandial group consisting of rats killed before feeding, 2) a prandial group consisting of rats killed during the meal, and 3) a postprandial group consisting of rats killed 8-12 min after the meal. Findings revealed site-specific changes in BN and CRF content during the course of a meal. During ingestion, levels of BN were significantly elevated at the paraventricular, arcuate, and dorsomedial nuclei of the hypothalamus and reduced at the nucleus accumbens. In the case of CRF, feeding-related alterations were observed at the lateral (LH) and ventromedial (VMH) hypothalamic nuclei and at the central nucleus of the amygdala (Ce). At the LH, CRF content decreased after feeding compared with preprandial levels. At the VMH, CRF levels were significantly elevated both before and after food intake compared with prandial levels. In contrast, at the Ce marked increases in CRF concentrations were observed during ingestion. These data demonstrate, for the first time, site-specific fluctuations of BN and CRF in relationship to the animal's feeding status and suggest that these peptides may play a role in the regulation of food intake.

Rapid alterations of hypothalamic and hippocampal bombesin-like peptide levels with feeding status.

It has been suggested that bombesin (BN)-like peptides may play a physiological role in the control of food intake. We studied the time course of changes in the levels of central BN-like peptides during a meal. Four groups of animals were used: rats that were food (but not water) deprived for 12-h period (preprandial group) and then given access to food for either 10 min (partially satiated group) or 35 min (postprandial group). The fourth group constituted nondeprived controls (ad libitum fed group). BN-like immunoreactivity (BLI) of the hypothalamus, hippocampus, and medulla was determined using a radioimmunoassay. Our data revealed that at the hypothalamus, the BLI content dropped significantly after food deprivation (preprandially), and returned to the ad libitum fed control levels after the meal (postprandially). At the hippocampus, food deprivation did not affect the BLI levels; however, food ingestion significantly elevated the BLI content within 35 min. The medullary BLI levels failed to alter in relation to the feeding status. The observed rapid alterations suggest that the hypothalamic response to food intake is satiety linked and hence lend support to the contention that BN-like peptides play a physiological role in the central regulation of ingestive behavior. The alterations noted at the hippocampus implicate physiological role of BN-like peptides in other meal-associated processes (such as memory).

Short- and long-term behavioral effects of neonatal exposure to bombesin.

Subcutaneous (sc) administration of the tetradecapeptide bombesin (BN) (1-10 mg/kg) elicited grooming in rat pups of 1-10 days of age and the magnitude of this response decreased as a function of age. The form of grooming induced was qualitatively different from that seen following central injection of BN to adult rats. Subchronic neonatal exposure to BN (5 or 10 mg/kg; sc, twice daily for the first 8 postnatal days) had no effect on subsequent adult behavior displayed under mildly stressful or novel conditions, in the open field or in an elevated plus maze. However, both saline and the high dose of BN (10 mg/kg) pretreatments increased adult sensitivity to central BN (0.1 micrograms; icv) as compared to noninjected but neonatally handled controls or those rats neonatally pretreated with the lower dose of BN (5 mg/kg). This was best demonstrated by increases in scratching activity at the 0.1-micrograms dose of icv BN. Neonatal pretreatments had no effect on later adult sensitivity to BN injected intraperitoneally (ip). These data indicate that BN receptors in the rat central nervous system are pharmacologically functional from an early stage in ontogeny. Systems utilizing BN-like peptides are, to a degree, plastic early in ontogeny and altered adult sensitivity to BN icv can be achieved via subchronic exposure to BN during infancy. Endogenous BN-based mechanisms did not appear to play a role in the development and/or expression of behavior(s) elicited under mildly stressful or novel conditions.

Neuropeptide Y and behaviour: effects of intrastriatal NPY on circling behaviour of rats.

Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family and consists of 36 amino acids, sharing sequence homologies with the putative gut hormone peptides YY and PP. NPY dose-dependently stimulates food intake when administered icv into the paraventricular hypothalamic nucleus of rats. Icv NPY has also been reported to decrease locomotor activity, rearing and grooming behaviour. Little behavioural research focusing on other unconditioned behaviours has been conducted. However, intrastriatal injections of NPY have been shown to increase dopamine (DA) turnover there. As unilateral manipulations of central DA result in turning away from the side of higher DA activity, it is of interest to evaluate the effects of intrastriatal NPY on this behaviour. Preliminary results indicate that NPY produces a significant contralateral turning bias when injected directly into the striatum. This raises the intriguing possibility that contralateral turning induced by intrastriatal NPY may be mediated by DA.

Protective effect of selenium on certain hepatotoxic and pancreotoxic manifestations of subacute cadmium administration.

Administration of cadmium chloride (1.0 mg/kg s.c.) to rats, twice a day for 7 days, significantly stimulated the activities of hepatic pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose 6-phosphatase, markedly increased the concentration of hepatic cyclic adenosine monophosphate and circulating blood glucose and significantly reduced serum insulin levels. Furthermore, subacute exposure to cadmium induced glucose intolerance that was associated with a decreased pancreatic secretory activity as evidenced by lowered insulinogenic indices and marked inhibition of phentolamine-stimulated insulin release. In contrast to cadmium, administration of selenium dioxide (2 X 1.0 mg/kg/day s.c., 7 days) failed to alter significantly the activities of gluconeogenic enzymes, hepatic cyclic adenosine monophosphate, blood glucose or serum insulin levels, glucose tolerance or the pancreatic secretory activity. However, administration of selenium concurrently with cadmium completely prevented the cadmium-induced increases of hepatic gluconeogenic enzymes. Treatment with selenium ameliorated the cadmium-induced hyperglycemia, hypoinsulinemia, glucose intolerance and the suppression of pancreatic secretory activity, whereas it failed to alter significantly the cadmium-induced elevation of hepatic cyclic AMP levels. Data provide evidence suggesting that subacute exposure to cadmium alters several parameters of carbohydrate metabolism and suppresses pancreatic secretory activity and that administration of selenium alone is without any appreciable effect on the above parameters. However, administration of selenium concurrently with cadmium prevents, to varying degrees, several of the cadmium-induced metabolic and functional changes.