The Anxiolytic Effect of Polysaccharides from Stellariae Radix through Monoamine Neurotransmitters, HPA Axis, and ECS/ERK/CREB/BDNF Signaling Pathway in Stress-induced Male Rats.

BACKGROUND: Stellaria dichotoma L. var. lanceolata Bge. is renowned for its efficacy in "clearing deficiency heat" and represents a significant traditional Chinese medicine (TCM) resource. Modern pharmacology has demonstrated the anti-anxiety effects of Stellaria dichotoma L. var. lanceolata Bge. polysaccharides (SDPs). SDPs are one of the active constituents of Stellaria dichotoma L. var. lanceolata Bge. This study presents the first extraction of SDPs and investigates their potential molecular mechanisms and anxiolytic effects that are not previously reported. METHODS: First, SDPs were obtained by water extraction and alcohol precipitation and analyzed for their monosaccharide composition by high performance liquid chromatography (HPLC). Male SD rats were subjected to a two-week indeterminate empty bottle stress procedure and a three-day acute restraint stress procedure, during which diazepam (DZP) (1 mg/kg) and SDPs (50, 100 and 200 mg/kg, intragastrically) were administered. A number of behavioral tests, including the elevated plus maze test (EPM), the open field test (OFT) and the light/dark box test (LDB), were used to assess the anti-anxiety potential of SDPs. Serum levels of Corticosterone (CORT) and Adrenocorticotropic hormone (ACTH), as well as the levels of Dopamine (DA) and serotonin (5-HT) found in the hippocampus and frontal cortex, were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits. In addition, protein levels of key proteins cAMP-response element binding protein (CREB), phospho-CREB (p-CREB), brain-derived neurotrophic factor (BDNF), ERK½, p-ERK½, and GAPDH expression in rat hippocampus were measured by Western blot analysis, and modulation of the endocannabinoid system was assessed by immunohistochemistry. RESULTS: Following administration of SDPs (50, 100, 200 mg/kg) and diazepam 1 mg/kg, anxiolytic activity was exhibited through an increase in the percentage of arm opening times and arm opening time of rats in the elevated plus maze. Additionally, there was an increase in the number of times and time spent in the open field center, percentage of time spent in the open box, and shuttle times in the LDB. Furthermore, tissue levels of DA and 5-HT were increased in the hippocampus and frontal cortex of rats after treatment with SDPs. In addition, SDPs significantly decreased serum levels of CORT and ACTH in rats. SDPs also effectively regulated the phosphorylation of the extracellular regulated protein kinases (ERK) and CREB-BDNF pathway in the hippocampus. Moreover, the expression levels of CB1 and CB2 proteins were heightened due to SDPs treatment in rats. CONCLUSIONS: The study verified that SDPs alleviate anxiety in the EBS and ARS. The neuroregulatory behavior is accomplished by regulating the Monoamine neurotransmitter, HPA axis, and ECB-ERK-CREB-BDNF signaling pathway.

Clove volatile oil-loaded nanoemulsion reduces the anxious-like behavior in adult zebrafish.

BACKGROUND: Clove volatile oil (CVO) and its major compound, eugenol (EUG), have anxiolytic effects, but their clinical use has been impaired due to their low bioavailability. Thus, their encapsulation in nanosystems can be an alternative to overcome these limitations. OBJECTIVES: This work aims to prepare, characterize and study the anxiolytic potential of CVO loaded-nanoemulsions (CVO-NE) against anxious-like behavior in adult zebrafish (Danio rerio). METHODS: The CVO-NE was prepared using Agaricus blazei Murill polysaccharides as stabilizing agent. The drug-excipient interactions were performed, as well as colloidal characterization of CVO-NE and empty nanoemulsion (B-NE). The acute toxicity and potential anxiolytic activity of CVO, EUG, CVO-NE and B-NE against adult zebrafish models were determined. RESULTS: CVO, EUG, CVO-NE and B-NE presented low acute toxicity, reduced the locomotor activity and anxious-like behavior of the zebrafish at 4 - 20 mg kg-1. CVO-NE reduced the anxious-like behavior of adult zebrafish without affecting their locomotor activity. In addition, it was demonstrated that anxiolytic activity of CVO, EUG and CVO-NE is linked to the involvement of GABAergic pathway. CONCLUSION: Therefore, this study demonstrates the anxiolytic effect of CVO, in addition to providing a new nanoformulation for its administration.

Production of biomolecules of interest to the anxiolytic herbal medicine industry in yellow passionfruit leaves (Passiflora edulis f. flavicarpa) promoted by mycorrhizal inoculation.

BACKGROUND: Our contemporary way of life has led us to consume high amounts of chemically-synthesized allopathic medicinal products and anxiolytics to which a viable alternative is the use of Passiflora-based herbal medicines with composition containing vitexin, a flavonoid with anxiolytic and antidepressant properties. Arbuscular mycorrhizal fungi (AMF) are known for enhancing the production of biomolecules, however, increase production of phytochemistry in Passiflora edulis f. flavicarpa has not been reported in the literature. Our aim was to select AMF to benefit the production of vitexin in leaves of P. edulis by inoculating seedlings in the region of roots with Acaulospora longula, Claroideoglomus etunicatum and Gigaspora albida. RESULTS: The inoculation increased the concentration of vitexin in 63.64% and the inoculation with A. longula also increased the content of flavonoids and total saponins in the leaves in relation to the control. CONCLUSION: The increase in the production of vitexin in the leaf in response to the inoculation with AMF, with emphasis to A. longula, interests the pharmaceutical industry and can generate profit to the production of yellow passionfruit-based anxiolytic herbal medicine. © 2019 Society of Chemical Industry.

Acupuncture modulates stress response by the mTOR signaling pathway in a rat post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a psychiatric disease that can form following exposure to a traumatic event. Acupuncture has been proposed as a beneficial treatment for PTSD, but the underlying mechanisms remain unclear. The present study investigated whether acupuncture improves depression- and anxiety-like behaviors induced using a single prolonged stress (SPS) as a PTSD rat model. In addition, we investigated whether the effects were mediated by increased mTOR activity and its downstream signaling components, which contribute to protein synthesis required for synaptic plasticity in the hippocampus. We found that acupuncture at HT8 significantly alleviated both depression- and anxiety-like behaviors induced by SPS in rats, as assessed by the forced swimming, elevated plus maze, and open field tests; this alleviation was blocked by rapamycin. The effects of acupuncture were equivalent to those exerted by fluoxetine. Acupuncture regulated protein translation in the mTOR signaling pathway and enhanced the activation of synaptic proteins, PSD95, Syn1, and GluR1 in the hippocampus. These results suggest that acupuncture exerts antidepressant and anxiolytic effects on PTSD-related symptoms by increasing protein synthesis required for synaptic plasticity via the mTOR pathway in the hippocampus. Acupuncture may be a promising treatment for patients with PTSD and play a role as an alternative PTSD treatment.

Drug interaction study of flavonoids toward CYP3A4 and their quantitative structure activity relationship (QSAR) analysis for predicting potential effects.

The high risk of herb-drug interactions (HDIs) mediated by the herbal medicines and dietary supplements which containing abundant flavonoids had become more and more frequent in our daily life. In our study, the inhibition activities of 44 different structures of flavonoids toward human CYPs were systemically evaluated for the first time. According to our results, a remarkable structure-dependent inhibition behavior toward CYP3A4 was observed in vitro. Some flavonoids such as licoflavone (12) and irilone (30) exhibited the selective inhibition toward CYP3 A4 rather than other major human CYPs. To illustrate the interaction mechanism, the inhibition kinetics of various compounds was further performed. Sophoranone (1), apigenin (10), baicalein (11), 5,4'-dihydroxy-3,6,7,8,3'-pentamethoxyflavone (15), myricetin (23) and kushenol K (38) remarkably inhibited the CYP3 A4-catalyzed bufalin 5'-hydroxylation reaction, with Ki values of 2.17 ±â€¯0.29, 6.15 ±â€¯0.39, 9.18 ±â€¯3.40, 2.30 ±â€¯0.36, 5.00 ±â€¯2.77 and 1.35 ±â€¯0.25 µM, respectively. Importantly, compounds 1, 11, 15, 23 and 38 could significantly inhibit the metabolism of some clinical drugs in vitro, and these drug-drug interactions (DDIs) of myricetin (23) or kushenol K (38) with clinical drug diazepam were further verified in human primary hepatocytes, respectively. Finally, a quantitative structure-activity relationship (QSAR) of flavonoids with their inhibitory effects toward CYP3 A4 was established using computational methods. Our findings illustrated the high risk of herb-drug interactions (HDIs) caused by flavonoids and revealed the vital structures requirement of natural flavonoids for the HDIs with clinical drugs eliminated by CYP3 A4. Our research provided the useful guidance to safely and rationally use herbal medicines and dietary supplements containing rich natural flavonoids components.

Effect of Benifuuki Tea on Cytochrome P450-mediated Metabolic Activity in Rats.

BACKGROUND/AIM: Benifuuki tea has recently been used as an alternative therapy for pollinosis, and it may be consumed with pharmaceutical drugs. This study aimed to examine cytochrome P450 (CYP)-mediated food-drug interactions with Benifuuki tea in rats. MATERIALS AND METHODS: The inhibitory effects of Benifuuki tea and (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me) on CYP activities were evaluated in vitro. Midazolam pharmacokinetics was investigated after two treatments with Benifuuki tea. In an ex vivo study, CYP activities were determined after 1-week-treatment with the tea. RESULTS: Benifuuki tea and EGCG3"Me inhibited CYP2D and CYP3A activities in a concentration-dependent manner in vitro. However, MDZ metabolism did not change by Benifuuki treatment in vivo and ex vivo. In contrast, CYP2D activity was decreased ex vivo. CONCLUSION: Normal intake of Benifuuki tea is not likely to cause food-drug interactions by CYP3A inhibition or induction. In contrast, Benifuuki tea consumption may lead to food-drug interactions through the inhibition of CYP2D.

Synthesis, Computational and Pharmacological Evaluation of N-(2-benzoyl- 4-chlorophenyl)-2-(4-(Substituted Phenyl) Piperazin-1-yl) Acetamides as CNS Agents.

BACKGROUND: A series of new N-(2-benzoyl-4-chlorophenyl)-2-(4-(substituted phenyl) piperazin-1-yl) acetamides (3a-j) have been synthesized by the chloroacetylation of 2-amino-5- chlorbenzophenone which was further reacted with substituted phenylpiperazine. MATERIAL: The chemical structures of the compounds were confirmed on the basis of their TLC, IR, 1HNMR, 13CNMR and by elemental analysis. The physicochemical similarity of the target compounds with respect to standard drug diazepam was assessed by calculating from a set of physicochemical properties using software programs. CONCLUSION: Molecular docking studies revealed that the target compounds correctly dock into the binding pocket of the GABAA receptor, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. The anxiolytic and skeletal muscle relaxant activity of the target compounds (3a-j) were evaluated in albino mice. Among them, compound 3h showed potent anxiolytic and skeletal muscle relaxant activity.

Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

BACKGROUND: It has recently been suggested that the adenosine A2A receptor plays a role in several animal models of depression. Additionally, A2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. METHODS: In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A2A receptors but poor A1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. RESULTS: The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. CONCLUSION: Available data support the proposition that the examined compounds with adenosine A2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents.

The first dipeptide ligand of translocator protein: Design and anxiolytic activity.

On the basis of the structure of Alpidem, a pyrazolopyrimidine ligand of the translocator protein (TSPO), a dipeptide TSPO ligand, N-carbobenzoxy-L-tryptophanyl-L-isoleucine amide (GD-23), was designed and synthesized using our own original peptide design strategy. This compound exhibited anxiolytic activity in BALB/cAnN mice in the "open-field" test and in outbred CD1 mice in the "elevated plus maze" test. The stereoselectivity of the anxiolytic effect of GD-23 is demonstrated. The results of this study suggest that GD-23 is a ligand of the translocator protein, and its structure can become the basis for creating anxiolytics with a fundamentally new mechanism of action.

GABAA Receptor Binding Assays of Standardized Leonurus cardiaca and Leonurus japonicus Extracts as Well as Their Isolated Constituents.

A main traditional use of European Leonurus cardiaca and East Asian Leonurus japonicus is in the treatment of neurological disorders such as anxiety, depression, nervousness, and as a sedative for insomnia. However, their mechanism of action is still under discussion. As anxiety and depressive disorders are increasingly being recognized as connected to dysfunctions of the gamma-aminobutyric acid system, the in vitro effects of standardized L. cardiaca and L japonicus extracts as well as five of their isolated constituents, namely, the labdane-type isoleosibirin, the novel iridoid 7R-chloro-6-desoxy-harpagide, the phenylethanoid lavandulifolioside, and the N-containing compounds stachydrine and leonurine, on this type of neuronal receptor were investigated for the first time. Extracts of L. cardiaca and L. japonicus, characterized by reversed-phase high-performance liquid chromatography determination, as well as their above named isolated, possible active constituents of different chemical nature were tested in several receptor binding assays at rat GABAA receptors using [(3)H]-SR95 531 and [(3)H]-Ro-15-1788 (flumazenil)/diazepam control. The L. cardiaca and L. japonicus extracts as well as leonurine inhibited the concentration-dependent binding of [(3)H]-SR95 531 to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor with a high binding affinity: IC50s 21 µg/ml, 46 µg/ml, and 15 µg/ml, respectively. In contrast, binding to the benzodiazepine site of the rat gamma-aminobutyric acid type A receptor had a 15 to 30 times lower binding affinity than to the gamma-aminobutyric acid site. The presented experiments provide hints that the neurological mechanism of action of L. cardiaca and L. japonicus may essentially be based on their interaction to the gamma-aminobutyric acid site of the gamma-aminobutyric acid type A receptor, while the benzodiazepine site most probably does not contribute to this effect. In the case of L. japonicus, these effects can be at least partially explained by its leonurine constituent, whereas the active principle of L. cardiaca, which does not contain leonurine, is subject to further research as none of the other investigated individual constituents displayed significant activity in the applied test system.

Cytoprotective Effect of Afobazole and Its Main Metabolite M-11.

Cell damage depending on activity of quinone reductase 2 (MT3 receptor) was simulated in experiments on bone marrow cell suspension and assessed by menadione-induced DNA breaks measured by comet assay. We analyzed the protective effect of afobazole interacting with MT1, MT3, σ1 receptors, and monoamine oxidase A and its main metabolite M11 that specifi cally binds to MT3 receptors. Both compounds reduced the level of menadione-induced DNA damage (afobazole was effective in lower concentrations in comparison with M-11). Conclusion was made on the contribution of MT3 receptors to the protective effect of afobazole, but the observed concentration differences indicate possible contribution of other targets of anxiolytic drug to the protective mechanisms.

[Mechanism on atrial natriuretic peptide receptor in anti-anxiety with acupuncture based on its tranquilizing effect].

Anxiety disorder is one of the most common mental disorders and seriously impairs the physical and mental health of patients. Due to the efficacy of acupuncture for tranquilization, acupuncture displays its unique advantage on the treatment of anxiety disorder, but the relevant biological mechanism has not been elaborated. The modern medicine study has proved that the heart and brain have their own independent natriuretic peptide (NP) system. The dysfunction of ANP and its receptor are closely related to the occurrence of anxiety disorder. The ANP acts on anti-anxiety. Hence, focusing on the three aspects, named the anti-anxiety effect of acupuncture based on its tranquilizing effect, the anti-anxiety effect of ANP and the positive regulation of acupuncture on NP, the mechanism on ANP and its receptor was explored in anti-anxiety with acupuncture based on tranquilizing effect, and the idea was put forward on that the anti-anxiety effect of acupuncture was possibly based on its action of tranquilization through regulating the ANP and its receptor. As a result, it is expected to provide the theoretic support for the mechanism study on anti-anxiety with acupuncture based on its tranquilizing effect.

5-HT(1A) receptor binding in the dorsal raphe nucleus is implicated in the anxiolytic-like effects of Cinnamomum cassia.

Previously we reported that the 50% EtOH extract of Cinnamomum cassia (C. cassia) possesses anxiolytic-like activity in the mouse elevated plus maze (EPM) test. This activity was blocked by the 5-HT(1A) receptor antagonist, WAY 100635. Therefore, in order to investigate the effect of C. cassia on 5-HT(1A) receptor binding, quantitative autoradiography of 5-HT(1A) receptors was carried out in brains of mice treated acutely and repeatedly with C. cassia. Binding of [(3)H]8-OH-DPAT to the 5-HT(1A) receptor was investigated in the mouse brain. After a single treatment of C. cassia (750 mg/kg, p.o.), [(3)H]8-OH-DPAT binding showed a significant increase in the dorsal raphe nucleus (DRN). After repeated treatment with C. cassia (100mg/kg, once a day for 5 days, p.o.), [(3)H]8-OH-DPAT binding showed no significant change in any brain region. Taken together, the anxiolytic-like effect of the 50% EtOH extract of C. cassia might be mediated by region specific change of 5-HT(1A) receptors in the dorsal raphe nucleus.

Fish oil supplementation of maternal rats on an n-3 fatty acid-deficient diet prevents depletion of maternal brain regional docosahexaenoic acid levels and has a postpartum anxiolytic effect.

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are the major polyunsaturated fatty acids (PUFA) in the neuronal membrane. Most DHA and AA accumulation in the brain occurs during the perinatal period via placenta and milk. This study examined whether maternal brain levels of DHA and AA are depleted during pregnancy and lactation due to meeting the high demand of the developing nervous system in the offspring and evaluated the effects of the reproductive cycle on serotonin metabolism and of fish oil (FO) on postpartum anxiety. Pregnant rats were fed during pregnancy and lactation with a sunflower oil-based n-3 PUFA-deficient diet without or with FO supplementation, which provided 0.37% of the energy source as n-3 PUFA, and the age-matched virgin rats were fed the same diets for 41 days. In both sets of postpartum rats, decreased DHA levels compared to those in virgin females were seen in the hypothalamus, hippocampus, frontal cortex, cerebellum, olfactory bulb and retina, while AA depletion was seen only in the hypothalamus, hippocampus and frontal cortex. Serotonin levels were decreased and turnover increased in the brainstem and frontal cortex in postpartum rats compared to virgin rats. FO supplementation during pregnancy and lactation prevented the decrease in maternal brain regional DHA levels, inhibited monoamine oxidase-A activity in the brainstem and decreased anxiety-like behavior. We propose that the reproductive cycle depletes maternal brain DHA levels and modulates maternal brain serotonin metabolism to cause postpartum anxiety and suggest that FO supplementation may be beneficial for postpartum anxiety in women on an n-3 PUFA-deficient diet.

Orally administered L-ornithine elevates brain L-ornithine levels and has an anxiolytic-like effect in mice.

Intracerebroventricular injection of L-ornithine has demonstrated sedative and hypnotic effects in neonatal chicks exposed to acute stressful conditions. However, whether orally administered L-ornithine can reduce acute mental stress remains to be defined. To clarify the nutritional importance of L-ornithine in controlling the stress response, in Experiment 1 we first investigated whether orally administered L-ornithine can be transported into the brain of mice. Mice were orally administered L-ornithine (3 mmol/water 10 ml/kg, per os). L-Ornithine levels were significantly elevated in the cerebral cortex and hippocampus at 30 and 60 minutes post-administration. In Experiment 2, the effect of orally administered L-ornithine (0, 0.1875, 0.75 and 3 mmol/water 10 ml/kg, per os) on anxiety-like behavior in mice exposed to the elevated plus-maze test was examined at 30 minutes post-administration. There was a significant increase in the percentage of time spent and entries in the open arms in the group receiving 0.75 mmol of L-ornithine compared to the control group. Furthermore, locomotion activity in a novel environment was not significantly changed between the control group and 0.75 mmol of L-ornithine group in Experiment 3. Therefore, it appears that orally administrated L-ornithine is bioavailable to the rodent brain and reduces anxiety-like behavior as demonstrated by the elevated plus-maze test.

L-Theanine: properties, synthesis and isolation from tea.

Theanine is a non-protein amino acid that occurs naturally in the tea plant (Camellia sinensis) and contributes to the favourable taste of tea. It is also associated with effects such as the enhancement of relaxation and the improvement of concentration and learning ability. It is also linked with health benefits including the prevention of certain cancers and cardiovascular disease, the promotion of weight loss and enhanced performance of the immune system. Thus, there has been a significant rise in the demand for theanine. While theanine has been chemically and biologically synthesised, techniques to isolate theanine from natural sources remain an important area of research. In this review article, the properties and health benefits of theanine are summarised and the synthesis and isolation of theanine are reviewed and discussed. Future perspectives for the isolation of theanine from natural sources are also outlined.

Pharmacological evaluation of Potentilla alba L. in mice: adaptogenic and central nervous system effects.

CONTEXT: Potentilla alba L. (Rosaceae) rhizomes have anti-inflammatory, antioxidant, and adaptogenic effects and are used for the treatment of diarrhea and intestinal colic. However, the data concerning the adaptogenic and central nervous system activities of P. alba are fragmentary. OBJECTIVES: To determine the effect of oral administration of dried P. alba extract on the swimming endurance, light/dark exploration, and open-field tests for mice. MATERIALS AND METHODS: The mice were orally administered Rhodiola rosea extract (RR group); dry extract of P. alba at doses of 12, 36, or 72 mg/kg (groups: PA12, PA36, and PA72); or distilled water (control group) for 7 consecutive days. RESULTS: The swimming times of the RR, PA36, and PA72 groups were significantly longer than those of the control group. The administration of P. alba significantly increased the light time, latency time, and the number of rearings in a dose-dependent manner. In the open-field test, the P. alba extract at a dose of 12 mg/kg produced a significant increase in the frequency of head dipping and the number of squares crossed and a significant decrease in grooming compared with the control treatment. CONCLUSION: The current findings demonstrate that P. alba extracts significantly increased swimming endurance time and have anxiolytic-like action with a predominant locomotor component.

Monoamine reuptake inhibition and mood-enhancing potential of a specified oregano extract.

A healthy, balanced diet is essential for both physical and mental well-being. Such a diet must include an adequate intake of micronutrients, essential fatty acids, amino acids and antioxidants. The monoamine neurotransmitters, serotonin, dopamine and noradrenaline, are derived from dietary amino acids and are involved in the modulation of mood, anxiety, cognition, sleep regulation and appetite. The capacity of nutritional interventions to elevate brain monoamine concentrations and, as a consequence, with the potential for mood enhancement, has not been extensively evaluated. The present study investigated an extract from oregano leaves, with a specified range of active constituents, identified via an unbiased, high-throughput screening programme. The oregano extract was demonstrated to inhibit the reuptake and degradation of the monoamine neurotransmitters in a dose-dependent manner, and microdialysis experiments in rats revealed an elevation of extracellular serotonin levels in the brain. Furthermore, following administration of oregano extract, behavioural responses were observed in mice that parallel the beneficial effects exhibited by monoamine-enhancing compounds when used in human subjects. In conclusion, these data show that an extract prepared from leaves of oregano, a major constituent of the Mediterranean diet, is brain-active, with moderate triple reuptake inhibitory activity, and exhibits positive behavioural effects in animal models. We postulate that such an extract may be effective in enhancing mental well-being in humans.

Bridging the gap between GPCR activation and behaviour: oxytocin and prolactin signalling in the hypothalamus.

Neuropeptides of the brain are important neuromodulators, controlling behaviour and physiology. They signal through G protein-coupled receptors (GPCR) that couple to complex intracellular signalling pathways. These signalling networks integrate information from multiple sources, resulting in appropriate physiological and behavioural responses to environmental and internal cues. This paper will focus on the neuropeptides oxytocin and prolactin with respect to (1) the regulation of neuroendocrine stress responses and anxiety, and (2) the receptor-mediated molecular mechanisms underlying these actions of the neuropeptides. Besides its significant reproductive functions when released into the bloodstream, brain oxytocin reduces the activity of the hypothalamo-pituitary-adrenal (HPA) axis as well as anxiety-related behaviour in male and female rats. Oxytocin mediates its anxiolytic effect, at least in part, via binding to its GPCR in the hypothalamic paraventricular nucleus, followed by transactivation of the epidermal growth factor receptor, and subsequent activation of a MEK-extracellular signal-regulated kinase (ERK) MAP kinase pathway. Prolactin, by binding to its GPCR receptors, of which there are short and long forms, also activates ERK, and this is necessary for the control of the expression of corticotrophin-releasing hormone-an important regulator of the HPA axis. Liganded oxytocin and prolactin may also recruit other signalling pathways, but how these pathways contribute to the observed behavioural and physiological effects remains to be established. GPCR-mediated oxytocin and prolactin neuronal signalling are illustrative of the complexity of GPCR-activated regulation of appropriate neuroendocrine and behavioural responses to environmental and physiological demands.

Central nervous system activities of two diterpenes isolated from Aloysia virgata.

Using the guide of a competitive assay for the benzodiazepine binding site in the γ-aminobutyric acid type A receptor (GABA(A)), two active diterpenes were isolated from the aerial parts of Aloysia virgata (Ruíz & Pavón) A.L. Jussieu var. platyphylla (Briquet) Moldenke. These compounds, identified as (16R)-16,17,18-trihydroxyphyllocladan-3-one (1) and (16R)-16,17-dihydroxyphyllocladan-3-one (2) on the basis of spectral data, competitively inhibited the binding of [(3)H]-FNZ to the benzodiazepine binding site with K(i)±S.E.M. values of 56±19 µM and 111±13 µM, respectively. The behavioral actions of these diterpenes, intraperitoneally (i.p.) administered in mice, were examined in the plus-maze, holeboard, locomotor activity and light/dark tests. Compound 1 exhibited anxiolytic-like effects in mice evidenced by a significant increase of the parameters measured in the holeboard test (the number of head dips at 0.3 mg/kg and 3 mg/kg, the rears at 1 mg/kg and the time spent head-dipping at 3 mg/kg), in the plus-maze assay (the percentage of open arm entries at 1 mg/kg) and in the light/dark test (the time in light and the number of transitions at 1 mg/kg). Compound 2 augmented the number of rearings in the holeboard apparatus (at 0.3 mg/kg and 1 mg/kg) and the locomotor activity (at 1 mg/kg). These results reveal the presence of neuroactive compounds in Aloysia virgata.