Characterization and pharmacokinetic evaluation of microcomposite particles of alpha lipoic acid/hydrogenated colza oil obtained in supercritical carbon dioxide.

The work reported here is an extension of our previous findings in which supercritical composite particles (SCP) of alpha lipoic acid (ALA) masked with hydrogenated colza oil (HCO) named as ALA/HCO/SCP were obtained by the modified particles from gas-saturated solutions (PGSS) process in supercritical carbon dioxide in order to obscure the unpleasant taste and odor of ALA. The masking effect on ALA/HCO/SCP was compared with the widely used mechano-chemically masked formulation of ALA and HCO named as MC-50F. In the present study, ALA/HCO/SCP particles were found to have a significant improvement in regard to bitterness, numbness, and smell compared to ALA bulk powders suggesting they were well coated. The pharmacokinetic parameters for ALA/HCO/SCP and ALA bulk powder gave similar values but were significantly different from those of MC-50F. The amount of ALA absorbed into the body, in the administered ALA/HCO/SCP, was comparable to that absorbed by ALA bulk powder, whereas about half portion of ALA of the MC-50F was not absorbed, because the ALA/HCO/SCP particles were small enough and the particles of MC-50F were relatively large and had smaller specific surface area. Therefore, this study suggested a newly masked candidate may offer functional particles with maintained efficacy.

Tokishakuyakusan ameliorates spatial memory deficits induced by ovariectomy combined with ß-amyloid in rats.

Previously, we reported that ovariectomy (OVX) combined with ß-amyloid peptide (Aß) impaired spatial memory by decreasing extracellular acetylcholine (ACh) levels in the dorsal hippocampus. Here, we investigated the effect of tokishakuyakusan (TSS), a Kampo medicine, on the impairment of spatial memory induced by OVX combined with Aß in rats. Repeated administration of TSS (300 mg/kg, p.o.) significantly decreased the number of errors in the eight-arm radial maze test. Though TSS had no effect on extracellular ACh levels at baseline, TSS significantly increased extracellular ACh levels in the dorsal hippocampus. These results suggest that TSS improves the impairment of spatial memory induced by OVX combined with Aß by (at least in part) increasing extracellular ACh levels in the dorsal hippocampus.

Goreisan Prevents Brain Edema after Cerebral Ischemic Stroke by Inhibiting Aquaporin 4 Upregulation in Mice.

BACKGROUND: Aquaporin 4 (AQP4) is a water-selective transport protein expressed in astrocytes throughout the central nervous system. AQP4 level increases after cerebral ischemia and results in ischemic brain edema. Brain edema markedly influences mortality and motor function by elevating intracranial pressure that leads to secondary brain damage. Therefore, AQP4 is an important target to improve brain edema after cerebral ischemia. The Japanese herbal Kampo medicine, goreisan, is known to inhibit AQP4 activity. Here, we investigated whether goreisan prevents induction of brain edema by cerebral ischemia via AQP4 using 4-hour middle cerebral artery occlusion (4h MCAO) mice. METHODS: Goreisan was orally administered at a dose of 500 mg/kg twice a day for 5 days before MCAO. AQP4 expression and motor coordination were measured by Western blotting and rotarod test, respectively. RESULTS: Brain water content of 4h MCAO mice was significantly increased at 24 hours after MCAO. Treatment with goreisan significantly decreased both brain water content and AQP4 expression in the ischemic brain at 24 hours after MCAO. In addition, treatment with goreisan alleviated motor coordination deficits at 24 hours after MCAO. CONCLUSIONS: The results of this study suggested that goreisan may be a useful new therapeutic option for ischemic brain edema.

Gas-saturated solution process to obtain microcomposite particles of alpha lipoic acid/hydrogenated colza oil in supercritical carbon dioxide.

Alpha lipoic acid (ALA), an active substance in anti-aging products and dietary supplements, need to be masked with an edible polymer to obscure its unpleasant taste. However, the high viscosity of the ALA molecules prevents them from forming microcomposites with masking materials even in supercritical carbon dioxide (scCO2). Therefore, the purpose of this study was to investigate and develop a novel production method for microcomposite particles for ALA in hydrogenated colza oil (HCO). Microcomposite particles of ALA/HCO were prepared by using a novel gas-saturated solution (PGSS) process in which the solid-dispersion method is used along with stepwise temperature control (PGSS-STC). Its high viscosity prevents the formation of microcomposites in the conventional PGSS process even under strong agitation. Here, we disperse the solid particles of ALA and HCO in scCO2 at low temperatures and change the temperature stepwise in order to mix the melted ALA and HCO in scCO2. As a result, a homogeneous dispersion of the droplets of ALA in melted HCO saturated with CO2 is obtained at high temperatures. After the rapid expansion of the saturated solution through a nozzle, microcomposite particles of ALA/HCO several micrometers in diameter are obtained.

Effects of Comb Tooth Cap Medicinal Mushroom, Hericium ramosum (Higher Basidiomycetes) Mycelia on DPPH Radical Scavenging Activity and Nerve Growth Factor Synthesis.

The goal of this study was to evaluate the antioxidant effects of and nerve growth factor (NGF) synthesis caused by Hericium ramosum mycelia. Wild mushroom fruiting bodies were collected from nature to isolate their mycelia. Pieces of H. ramosum fruiting bodies were plated onto 90-mm Petri dishes with potato dextrose agar medium to isolate their mycelia. Antioxidant activity was measured using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity in vitro; the ethanol extract from H. ramosum mycelia (63.11 µmol Trolox/g) was more potent than that of other mushroom mycelia extracts. There was a proportional relationship (R2 = 0.7929) between DPPH radical scavenging activity and total phenolic content in extracts of different mushroom mycelia. We investigated the ability of H. ramosum mycelia to inducing NGF synthesis in vivo. Oral administration of H. ramosum mycelia significantly increased concentrations of NGF in the hippocampus of intact mice. These results are the first concerning antioxidant activity and NGF synthesis of H. ramosum mycelia. These mushroom mycelia could be useful as food and/or nutritional supplements because of certain biological functions.

Cholinergic involvement and synaptic dynamin 1 expression in Yokukansan-mediated improvement of spatial memory in a rat model of early Alzheimer's disease.

The aim of this study was to investigate the effect of Yokukansan (YKS) on the impairment of spatial memory and cholinergic involvement in a rat model of early-phase Alzheimer's disease (AD). In this model, rats underwent four-vessel transient cerebral ischemia and then were treated with beta amyloid oligomers injected intracerebroventricularly once daily for 7 days. These animals showed memory impairment in an eight-arm radial maze task without histological evidence of apoptosis but with a decrease in expression of hippocampal dynamin 1, an important factor in synaptic vesicle endocytosis. Oral administration of YKS for 2 weeks significantly increased the number of correct choices and decreased the number of error choices in the eight-arm radial maze task (P < 0.05). Moreover, YKS significantly increased high K⁺-evoked potentiation of acetylcholine (ACh) release (P < 0.05) and significantly increased the expression of dynamin 1 (P < 0.01) in the hippocampus. The ameliorative effect of YKS on spatial memory impairment in our rat model of early-phase AD may be mediated in part by an increase in ACh release and modulation of dynamin 1 expression, leading to improved synaptic function. Future studies will determine whether YKS is similarly useful in the treatment of memory defects in patients diagnosed with early-stage AD.

Magnesium and the inflammatory response: potential pathophysiological implications in the management of patients with aneurysmal subarachnoid hemorrhage?

Cerebral vasospasm and delayed cerebral ischemia remain an unsolved problem in patients with aneurysmal subarachnoid hemorrhage (SAH). In theory, high-dose magnesium sulfate (MgSO(4)) therapy offers vascular and neuroprotective benefits and is therefore currently under evaluation. The intensity of the inflammatory response after SAH is associated with the outcome. The aim of the current study was to evaluate a possible link between the inflammatory response and MgSO(4) therapy, since magnesium (Mg(2+)) has anti-inflammatory properties. In 15 patients with SAH, inflammatory cytokine levels in the cerebrospinal fluid (CSF) and peripheral blood were determined daily using an enzyme-linked immunosorbent assay between day 4 and day 12. Eight patients were treated with standard therapy alone (group 1) and seven patients were treated with an additional, high-dose of MgSO(4) (group 2). Serum Mg(2+) levels in group 2 were significantly higher compared to group 1: 1.48 ± 0.04 mmol/L versus 0.90 ± 0.01 mmol/L, ρ<0.001. Interleukin-6 (IL-6) in the CSF was significantly lower in group 2 compared to group 1: 6680 ± 989 vs.11079 ± 1277 pg/mL, ρ = 0.021. A trend towards lower systemic IL-6 levels was found in group 2: 58 ± 7 versus 104 ± 21 pg/mL, ρ = 0.052. Systemic IL-1ß levels were significantly lower in group 2: 0.66 ± 0.11 and 0.15 ± 0.01 pg/mL (ρ<0.001), while the CSF levels did not differ. Tumor necrosis factor-α levels did not differ between the two groups. Although there were more patients with favorable outcome in group 2, the difference was not statistically significant. This was probably due to the small sample size. The results indicate a suppression of inflammatory cytokine release, in particular IL-6, in patients treated with high-dose MgSO(4). These results call for further studies of the effect of Mg(2+) on the inflammatory signaling pathway with regard to delayed cerebral ischemia following SAH.

Hypothalamic 2-arachidonoylglycerol regulates multistage process of high-fat diet preferences.

BACKGROUND: In this study, we examined alterations in the hypothalamic reward system related to high-fat diet (HFD) preferences. We previously reported that hypothalamic 2-arachidonoylglycerol (2-AG) and glial fibrillary acid protein (GFAP) were increased after conditioning to the rewarding properties of a HFD. Here, we hypothesized that increased 2-AG influences the hypothalamic reward system. METHODS: The conditioned place preference test (CPP test) was used to evaluate HFD preferences. Hypothalamic 2-AG was quantified by gas chromatography-mass spectrometry. The expression of GFAP was examined by immunostaining and western blotting. RESULTS: Consumption of a HFD over either 3 or 7 days increased HFD preferences and transiently increased hypothalamic 2-AG levels. HFD consumption over 14 days similarly increased HFD preferences but elicited a long-lasting increase in hypothalamic 2-AG and GFAP levels. The cannabinoid 1 receptor antagonist O-2050 reduced preferences for HFDs after 3, 7, or 14 days of HFD consumption and reduced expression of GFAP after 14 days of HFD consumption. The astrocyte metabolic inhibitor Fluorocitrate blocked HFD preferences after 14 days of HFD consumption. CONCLUSIONS: High levels of 2-AG appear to induce HFD preferences, and activate hypothalamic astrocytes via the cannabinoid system. We propose that there may be two distinct stages in the development of HFD preferences. The induction stage involves a transient increase in 2-AG, whereas the maintenance stage involves a long lasting increase in 2-AG levels and activation of astrocytes. Accordingly, hypothalamic 2-AG may influence the development of HFD preferences.

Yokukansan enhances pentobarbital-induced sleep in socially isolated mice: possible involvement of GABA(A)-benzodiazepine receptor complex.

In the present study, we investigated the effect of the Kampo medicine Yokukansan (YKS) on pentobarbital-induced sleep in group-housed and socially isolated mice. Socially isolated mice showed shorter sleeping time than the group-housed mice. YKS (300 mg/kg, p.o.) prolonged the pentobarbital-induced sleeping time in socially isolated mice without affecting pentobarbital sleep in group-housed mice. The prolongation of sleeping time by YKS was reversed by bicuculline (3 mg/kg, i.p.) and flumazenil (3 mg/kg, i.p.), but not WAY100635. These findings suggest that the GABA(A)-benzodiazepine receptor complex, but not 5-HT(1A) receptors, is involved in the reversal effect of YKS on the decrease of pentobarbital sleep by social isolation.

Yokukansan Enhances Pentobarbital-Induced Sleep in Socially Isolated Mice: Possible Involvement of GABA(A) - Benzodiazepine Receptor Complex.

In the present study, we investigated the effect of the Kampo medicine Yokukansan (YKS) on pentobarbital-induced sleep in group-housed and socially isolated mice. Socially isolated mice showed shorter sleeping time than the group-housed mice. YKS (300 mg/kg, p.o.) prolonged the pentobarbital-induced sleeping time in socially isolated mice without affecting pentobarbital sleep in group-housed mice. The prolongation of sleeping time by YKS was reversed by bicuculline (3 mg/kg, i.p.) and flumazenil (3 mg/kg, i.p.), but not WAY100635. These findings suggest that the GABA(A) - benzodiazepine receptor complex, but not 5-HT(1A) receptors, is involved in the reversal effect of YKS on the decrease of pentobarbital sleep by social isolation.

Effects of yokukansan on anxiety-like behavior in a rat model of cerebrovascular dementia.

Behavioral and psychological symptoms of dementia (BPSD) are commonly seen in patients with dementia. Current pharmacological approaches to treatment are inadequate, despite the availability of serotonergic agents to ameliorate anxiety, one of the symptoms of BPSD. The herbal medicine yokukansan has been demonstrated to improve BPSD in a randomized, single-blinded, placebo-controlled study. However, the mechanisms of the anxiolytic effect of yokukansan have not been clarified. There are also no reports on the anxiolytic effect of yokukansan in cerebrovascular ischemia models. In this study, we examined whether rats subjected to repeated cerebral ischemia exhibited anxiety-like behavior in a plus-maze task, a light/dark box test and an open-field task. We then investigated the effect of yokukansan on anxiety-like behavior in ischemic rats. Repeated ischemia was induced by the four-vessel occlusion method in which a 10-min ischemic episode was repeated once after 60 min. Yokukansan was orally administered once a day for 14 days from 7 days before ischemia induction. The last administration was performed 1 h before the behavioral experiments. The ischemic rats showed anxiety-like behavior in all three tasks, suggesting that this rat may be a good model for anxiety in cerebrovascular dementia. Yokukansan exhibited anxiolytic effects on the anxiety-like behavior in rats subjected to repeated cerebral ischemia, and exerted antagonistic effects on the wet-dog shakes induced by 1-(2,5-dimethoxy-4-indophenyl)-2-amino propane, a serotonin receptor (5-HT(2A)) agonist. This study revealed that yokukansan shows anxiolytic effects not only in normal animals but also in cerebrovascular model rats.

Increment of hypothalamic 2-arachidonoylglycerol induces the preference for a high-fat diet via activation of cannabinoid 1 receptors.

The aim of the present study is to examine the relationship between preference for HFD and 2-arachidonoylglycerol (2-AG), endogenous cannabinoid. The 3-day HFD intake induced preference for HFD, which was suppressed by CB1 antagonist, O-2050. Moreover, hypothalamic 2-AG was increased after 3-day HFD intake. Our results show that preference for HFD is induced by activation of CB1 receptors via an increment of 2-AG in hypothalamus.

Depression-like behavior and reduced plasma testosterone levels in the senescence-accelerated mouse.

During aging, levels of testosterone gradually decline in men and low levels of testosterone in aged men are accompanied by increased incidence of depressive disorders. The senescence-accelerated-prone mouse 10 (SAMP10) is well known as an animal model of aging. The purpose of this study was to investigate the motor function, anxiety levels, depression-related emotional responses, attentional function and plasma levels of testosterone and dehydroepiandrosterone (DHEA) in SAMP10. SAMP10 exhibited a significant prolongation of immobility time compared to that of the aged-matched control senescence-accelerated-resistant mouse 1 (SAMR1) in the tail suspension test for measuring depression. Moreover, significant low levels of plasma testosterone but not DHEA were found in SAMP10, and the testosterone levels were inversely correlated with the depression-like behavior. By contrast, we did not observe any significant differences between SAMP10 and SAMR1 in the open-field, rota-rod, elevated plus-maze, marble-burying behavior, or prepulse inhibition test. The results of the present study indicate that testosterone may play an important role in the depression-like behavior in SAMP10.

Ameliorating effects of Kangen-karyu on neuronal damage in rats subjected to repeated cerebral ischemia.

We previously reported that 21-day (14-day pre-ischemic and 7-day post-ischemic) treatment with Kangen-karyu (KGKR) improved spatial memory impairment and hippocampal neuronal death induced by repeated cerebral ischemia (2 x 10-min, 1-h interval) in rats. In the present study, we examined the effect of single and 14-day pre-ischemic KGKR treatment on neuronal damage in the same repeated cerebral ischemia model. Additionally, to determine the mechanisms of neuroprotection by KGKR at glutamatergic neurons, we examined the effects of KGKR on glutamate release induced by repeated cerebral ischemia in vivo, and on cell damage induced by both glutamate and kainate in primary cultured hippocampal neurons in vitro. The 14-day pre-ischemic KGKR (300 mg/kg, oral administration (p.o.)) treatment reduced neuronal damage and astrocyte expression induced by repeated cerebral ischemia. No effect was observed after single pre-ischemic KGKR treatment. Both single and 14-day KGKR treatment decreased glutamate release in the hippocampal CA1 region in intact rats; however, neither pre-ischemic KGKR treatment altered glutamate release during cerebral ischemia. In vitro, KGKR (100-1000 microg/mL) dose-dependently suppressed hippocampal neuronal damage induced by both glutamate (100 microM) and kainate (1 mM). These data suggest neuroprotection with KGKR requires continuous pre-ischemic treatment, and that the mechanisms of protection may be involved in inhibiting the glutamatergic receptors of the post-synaptic neurons.

The cannabinoid 1-receptor silent antagonist O-2050 attenuates preference for high-fat diet and activated astrocytes in mice.

Endocannabinoids have been shown to activate reward-related feeding and to promote astrocytic differentiation. We investigated whether high-fat diet (HFD) intake produced a preference for HFD via an endocannabinoid-dependent mechanism. In the conditioned place preference test, the 2-week HFD-intake group showed preference for HFD and had increased expression of a marker for reactive astrocytes, glial fibrillary acid protein (GFAP), in the hypothalamus. The cannabinoid CB(1)-receptor antagonist O-2050 reduced the preference for HFD and expression of GFAP in the hypothalamus. These results suggested that HFD intake led to the development of a preference for HFD via astrocytic CB(1) receptors in the hypothalamus.

Therapeutic Potential of Non-Psychotropic Cannabidiol in Ischemic Stroke.

Cannabis contains the psychoactive component delta8-tetrahydrocannabinol (delta8-THC), and the non-psychoactive components cannabidiol (CBD), cannabinol, and cannabigerol. It is well-known that delta8-THC and other cannabinoid CB1 receptor agonists are neuroprotective during global and focal ischemic injury. Additionally, delta8-THC also mediates psychological effects through the activation of the CB1 receptor in the central nervous system. In addition to the CB1 receptor agonists, cannabis also contains therapeutically active components which are CB1 receptor independent. Of the CB1 receptor-independent cannabis, the most important is CBD. In the past five years, an increasing number of publications have focused on the discovery of the anti-inflammatory, anti-oxidant, and neuroprotective effects of CBD. In particular, CBD exerts positive pharmacological effects in ischemic stroke and other chronic diseases, including Parkinson's disease, Alzheimer's disease, and rheumatoid arthritis. The cerebroprotective action of CBD is CB1 receptor-independent, long-lasting, and has potent anti-oxidant activity. Importantly, CBD use does not lead to tolerance. In this review, we will discuss the therapeutic possibility of CBD as a cerebroprotective agent, highlighting recent pharmacological advances, novel mechanisms, and therapeutic time window of CBD in ischemic stroke.

Neuroprotective effects of Kangen-karyu on spatial memory impairment in an 8-arm radial maze and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia in rats.

In the present study, we investigated the neuroprotective effects of Kangen-karyu (KGK) in a repeated cerebral ischemia model (2 x 10 min, 1-h interval). A 21-day pre- and post-ischemic treatment with KGK (10 - 300 mg/kg) and aspirin (5 mg/kg) improved the spatial memory impairment and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia. However, a 7-day post-ischemic treatment with KGK did not attenuate the spatial memory impairment and neuronal death in this model. To determine the mechanism of action of KGK, we investigated the effects of a 14-day pre-ischemic treatment with KGK on cerebral blood flow in the hippocampal area of the repeated cerebral ischemia model using laser Doppler flowmetry. The 14-day pre-ischemic treatment with KGK increased the cerebral blood flow during reperfusion. These results suggest that a 21-day pre- and post-ischemic treatment with KGK can protect against brain damage caused by cerebral ischemia by increasing the cerebral blood flow in the hippocampal area.

Yokukansan inhibits social isolation-induced aggression and methamphetamine-induced hyperlocomotion in rodents.

Behavioral and psychological symptoms of dementia (BPSD) are commonly seen in patients with Alzheimer's disease (AD) and other forms of senile dementia. BPSD have a serious impact on the quality of life of dementia patients, as well as on that of their caregivers. However, effective drug therapy for BPSD has not been established. Recently, the traditional Japanese medicine Yokukansan (YKS, Yi-gan san in Chinese) has been reported to improve BPSD, such as aggression, agitation, irritability, and hallucinations, in a randomized, single-blind, placebo-controlled study. However, the psychopharmacologic effects of YKS remain unexplored. In the present study, we investigated the effects of YKS on social isolation-induced aggressive behavior and methamphetamine- or MK-801-induced hyperlocomotion in rodents. Social isolation markedly induced aggressive behavior in male Wistar rats. Quetiapine at a dose of 10 mg/kg (per os (p.o.)) significantly inhibited this social isolation-induced aggressive behavior. YKS (100, 300 mg/kg, p.o.) also significantly inhibited the aggressive behavior. Moreover, risperidone (0.1 mg/kg, p.o.) significantly inhibited methamphetamine- or MK-801-induced hyperlocomotion in mice. YKS (300 mg/kg, p.o.) inhibited methamphetamine-induced hyperlocomotion, while YKS at the same dose had no effect on MK-801-induced hyperlocomotion. These findings suggest that YKS may be useful for the treatment of aggression and agitation, and that the psychopharmacologic effects of YKS might be mediated, in part, by inhibiting the activity of the dopaminergic system.