Masahiro Tabuchi

Ameliorative effects of yokukansan, a traditional Japanese medicine, on learning and non-cognitive disturbances in the Tg2576 mouse model of Alzheimer's disease.

AIM OF THIS STUDY Aim of the present study is to clarify the effects of yokukansan (TJ-54) on learning and non-cognitive disturbances in the Tg2576 mouse expressing the human form of the APP695SWE (APP-Tg mice), which is considered to be an animal model of Alzheimers disease. MATERIALS AND METHODS Powdered diets containing 0.5 and 1.0% TJ-54 were given to the mice for 10 months (from 5 to 15 months old). The Morris water-maze test, elevated plus-maze test, and open-field test were performed for evaluation of learning and non-cognitive disturbances. RESULTS Treatment with 1.0% TJ-54 for 5 months shortened the time it took for APP-Tg positive (+) mice to reach the platform in the Morris water-maze test. In the elevated plus-maze test, treatment with 1.0% TJ-54 for 2 months significantly reduced the increased number of entries and the time spent in open arms observed in APP-Tg(+) mice. In an open-field test, treatment of 1.0% TJ-54 for 9 months significantly suppressed the increase in locomotion observed in APP-Tg(+) mice. CONCLUSION These results suggest the possibility that TJ-54 ameliorates learning deficits and non-cognitive defects including a decrease in the anxiety (or disinhibition) and an increase in locomotor activity (hyperactivity) observed in APP-Tg(+) mice.

The blood-brain barrier permeability of geissoschizine methyl ether in Uncaria hook, a galenical constituent of the traditional Japanese medicine yokukansan.

Geissoschizine methyl ether (GM) in Uncaria hook, a galenical constituent of yokukansan is thought to be one of active components in the psychotropic effect of yokukansan, a traditional Japanese medicine (kampo medicine). However, there is no data on the blood–brain barrier (BBB) permeability of Uncaria hook-derived alkaloids containing GM. In this study, we investigated the BBB permeability of seven Uncaria hook alkaloids (GM, isocorynoxeine, isorhynchophylline, hirsuteine, hirsutine, rhynchophylline, and corynoxeine) using in vivo and in vitro methods. In the in vivo experiment, seven alkaloids in the plasma and brain of rats orally administered with yokukansan were measured by liquid chromatography–mass spectroscopy/mass spectrometric multiple reaction monitoring assay. In the in vitro experiment, the BBB permeability of seven alkaloids were examined using the BBB model composed of co-culture of endothelial cells, pericytes, and astrocytes. In the in vivo study, six components containing GM but not isocorynoxeine were detected in the plasma, and three (GM, hirsuteine, and corynoxeine) of components were detected in the brain. The in vitro BBB permeability data indicated that seven alkaloids were able to cross brain endothelial cells in culture conditions and that the BBB permeability of GM was higher than those of the other six alkaloids. These results suggest that target ingredient GM in yokukansan administered orally is absorbed into the blood and then reaches the brain through the BBB. This evidence further supports the possibility that GM is an active component in the psychotropic effect of yokukansan.

Simultaneous quantitative analyses of indole and oxindole alkaloids of Uncaria Hook in rat plasma and brain after oral administration of the traditional Japanese medicine Yokukansan using high-performance liquid chromatography with tandem mass spectrometry

Uncaria Hook (UH) alkaloids are involved in the beneficial effects of Yokukansan. However, the pharmacokinetics of UH alkaloids after oral administration of Yokukansan has not yet been sufficiently investigated. Therefore, we developed and validated a sensitive and specific high-performance liquid chromatography with tandem mass spectrometry (LC/MS/MS) method for the simultaneous quantitation of seven UH alkaloids (corynoxeine, isocorynoxeine, rhynchophylline, isorhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether) in rat plasma and brain. After protein precipitation with acetonitrile, chromatographic separation was performed using an Ascentis Express RP-amide column, with gradient elution with 0.2% formic acid and acetonitrile at 0.3 mL/min. All analytes in the plasma and brain showed good linearity over a wide concentration range (r > 0.995). Intra-day and inter-day variations of each constituent were 8.6 and 8.0% or less in the plasma, and 14.9 and 15.0% or less in the brain, respectively. The validated LC/MS/MS method was applied in the pharmacokinetic studies of UH alkaloids after oral administration of Yokukansan to rats. In the plasma, rhynchophylline, hirsutine, hirsuteine and geissoschizine methyl ether were detected, but only geissoschizine methyl ether was detected in the brain. These results suggest that geissoschizine methyl ether is an important constituent of the pharmacological effects of Yokukansan.

Glycyrrhiza and Uncaria Hook contribute to protective effect of traditional Japanese medicine yokukansan against amyloid β oligomer-induced neuronal death.

ETHNOPHARMACOLOGICAL RELEVANCE Yokukansan, a traditional Japanese (Kampo) medicine, composed of seven medicinal herbs has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Recently, this medicine has been reported to improve the behavioral and psychological symptoms of dementia (BPSD) that often become problematic in patients with Alzheimers disease (AD). AIM OF THE STUDY Amyloid β (Aβ) oligomers, which are extremely toxic to neurons, are involved in neurodegeneration in AD. In animals, yokukansan has been proven to improve memory impairments and BPSD-like behavior in transgenic mice overexpressing amyloid precursor protein and mice intracerebroventricularly injected with Aβ oligomers. These results suggest that yokukansan is potentially able to reduce the neurotoxicity of Aβ oligomers. Therefore, the present study aimed to explore the improving effects brought by yokukansan that consists of seven herbs for Aβ oligomer-induced neurotoxicity in vitro and to identify the candidate herbs in yokukansans action. MATERIALS AND METHODS Primary cultured rat cortical neurons were used. Neurotoxicity induced by Aβ oligomers (3µM) and improving effects of yokukansan (300-1000 µg/mL) and its constituent herbs were evaluated in MTT assay, DNA fragmentation analysis, and electron microscopic analysis at 48h after treatment with Aβ oligomers and drugs. Moreover, changes in expression of genes related to endoplasmic reticulum (ER) stress and in caspase-3 activity that is the enzyme closely related to apoptosis were analyzed to investigate the underlying mechanisms. RESULTS Yokukansan ameliorated Aβ oligomer-induced neuronal damage in a dose-dependent manner in the MTT assay. This drug also suppressed DNA fragmentation caused by Aβ oligomers. Electron microscopic analysis suggested that yokukansan reduced karyopyknosis and the expansion of rough ER caused by Aβ oligomers. However, neither Aβ oligomers nor yokukansan affected the mRNA expression of any ER stress-related genes, including CHOP and GRP78. On the other hand, yokukansan dose-dependently suppressed Aβ oligomer-induced activation of caspase-3. Among the seven constituents of yokukansan, Glycyrrhiza and Uncaria Hook (60-200 µg/mL) suppressed Aβ oligomer-induced neuronal damage, DNA fragmentation, karyopyknosis, and caspase-3 activation to almost the same extent as yokukansan. CONCLUSIONS The present results suggest that yokukansan possesses an ameliorative effect against Aβ oligomer-induced neuronal apoptosis through the suppression of caspase-3 activation. Glycyrrhiza and Uncaria Hook may, at least in part, contribute to the neuroprotective effect of yokukansan. These mechanisms may underlie the improving effects of yokukansan on memory impairment and BPSD-like behaviors induced by Aβ oligomers.

Effects of yokukansan and donepezil on learning disturbance and aggressiveness induced by intracerebroventricular injection of amyloid β protein in mice

The effects of yokukansan and donepezil on learning disturbance and aggressiveness were examined in amyloid β protein (Aβ)‐injected mice. Intellicage tests showed that both yokukansan and donepezil ameliorated Aβ‐induced learning disturbance, but the ameliorating effect of donepezil was not enhanced by concomitant administration of yokukansan. On the other hand, a social interaction test showed that Aβ‐induced aggressiveness was ameliorated by yokukansan, but not by donepezil. Co‐administration of both drugs also ameliorated aggressiveness, as did yokukansan alone. In vitro binding assays revealed that yokukansan did not bind to choline receptors or transporters. In vitro enzyme assays revealed that yokukansan did not affect choline acetyltransferase activity or inhibit acetylcholinesterase activity, as did donepezil. These results suggest that yokukansan might ameliorate aggressiveness without interfering with the pharmacological efficacy (antidementia effect) of donepezil and also that concomitant administration of yokukansan might be useful for amelioration of aggressiveness, which was not lessened by donepezil. The difference in the efficacies of both drugs may be due to a difference in their pharmacological mechanisms. Copyright

Protective effects of glycycoumarin and procyanidin B1, active components of traditional Japanese medicine yokukansan, on amyloid β oligomer-induced neuronal death

ETHNOPHARMACOLOGICAL RELEVANCE Yokukansan, a traditional Japanese (Kampo) medicine, is composed of seven medicinal herbs, and has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Yokukansan and its constituent herbs, Glycyrrhiza and Uncaria Hook, have recently been shown to have protective effects against amyloid β (Aβ) oligomer-induced apoptosis by suppressing the activation of caspase-3 in primary cultured neurons. The aim of the present study was to identify the effective components of Glycyrrhiza and Uncaria Hook against Aβ oligomer-induced neurotoxicity. We also attempted to clarify the mechanisms by which yokukansan and these herbs, as well as their components, suppressed the activation of caspase-3 in Aβ oligomer-treated neurons. MATERIALS AND METHODS Rat primary cultured cortical neurons were treated with Aβ oligomer (3 μM). The protective effects of 16 components derived from Glycyrrhiza or Uncaria Hook against Aβ oligomer-induced neurotoxicity were determined using the MTT reduction assay 48 h after the treatment. The suppressive effects of the test substances, i.e., yokukansan, Glycyrrhiza, Uncaria Hook, and screened components, on the Aβ oligomer-induced activation of caspase-3(/7) were evaluated using the caspase-Glo assay 48 h after the Aβ oligomer treatment. The suppressive effects of the test substances on the activation of caspase-8 and -9, both of which are located upstream of caspase-3, were also examined 24h after the Aβ oligomer treatment. RESULTS Two of the 16 components tested, glycycoumarin derived from Glycyrrhiza and procyanidin B1 derived from Uncaria Hook, significantly inhibited Aβ oligomer-induced neuronal death in a dose-dependent manner. Glycyrrhiza, Uncaria Hook, and yokukansan significantly suppressed the Aβ oligomer-induced activation of caspase-3 as well as caspase-8 and -9. Glycycoumarin also suppressed the activation of caspase-3, but not caspase-8 and -9. Procyanidin B1 suppressed the activation of caspase-3, -8, and -9. CONCLUSIONS Our results demonstrated that glycycoumarin and procyanidin B1 had ameliorative effects on Aβ oligomer-induced neurotoxicity. The neuroprotective effects of glycycoumarin are thought to be due to the attenuated activation of caspase-3, but not caspase-8 or -9. Procyanidin B1, as well as yokukansan, Glycyrrhiza, and Uncaria Hook, may attenuate the activation of caspase-3 by inhibiting that of caspase-8 and -9.

Histochemistry of acetylcholine receptors and acetylcholinesterase during the formation of neuromuscular junctions in the urodele Hynobius nigrescens

The development and structure of neuromuscular junctions (n‐m‐js) in stylopodia of forelimbs of larvae and adults of Hynobius nigrescens were histochemically investigated for acetylcholine receptors (AChRs) and acetylcholinesterase (AChE) activity. In larvae, the tetramethyl rhodamine‐labelled α‐bungarotoxin (TMR‐αBT) positive areas appeared either as small fluorescent spots or fluorescent plates of various sizes. The mature fluorescent plate was found to be formed by the successive addition of spots, and the plates thus established were arranged linearly parallel to the axes of muscle fibers. AChE activity occurred almost exactly at TMR‐αBT‐positive sites. In adults, plate assemblies were often seen as a single dotted line (type A form) for both AChR binding and AChE reaction, in contrast to larval n‐m‐js in which AChE activity appeared as a continuous line. By applying the TMR‐αBT method, two other forms of adult n‐m‐js were observed: type B, a long dotted line several plates wide; and type C, with a cluster of plates randomly dispersed over the whole width of the muscle fiber. It seems that protoforms of the latter two forms of n‐m‐js appear in the muscles just before and after metamorphosis.

Yokukansan, a Traditional Japanese Medicine, Enhances the L-DOPA-Induced Rotational Response in 6-Hydroxydopamine-Lesioned Rats: Possible Inhibition of COMT.

The aim of the present study was to investigate the effects of the traditional Japanese medicine yokukansan (YKS) on the function of dopamine (DA) in the rat nigrostriatal system. Unilateral 6-hydroxydopamine lesions were produced in the rat nigrostriatal system. Despite a marked loss in the striatal immunoreactivity of tyrosine hydroxylase on the lesion side, striatal serotonin (5-HT) immunoreactivity was not affected. Treatment using L-3,4-dihydroxyphenylalanine (L-DOPA) in conjunction with benserazide for 15 d induced abnormal involuntary movements (AIMs) such as locomotive (rotational response), axial, forelimb, and orolingual movements in the lesioned rats. The L-DOPA-induced locomotive and axial, but not forelimb and orolingual, AIMs were significantly increased and prolonged by the pre-administration of YKS. We next investigated the effects of YKS on the production of DA from L-DOPA in 5-HT synthetic RIN 14B cells. RIN 14B cells produced DA and its metabolite, 3-methoxytyramine (3-MT), following L-DOPA treatment. YKS significantly augmented DA production and inhibited its metabolism to 3-MT in a manner similar to the catechol-O-methyltransferase (COMT) inhibitor entacapone. YKS and some alkaloids (corynoxeine: CX, geissoschizine methyl ether: GM) in Uncaria hook, a constituent herb of YKS, also inhibited COMT activity, indicating that the augmenting effect of YKS on L-DOPA-induced DA production in 5-HT synthetic cells was due to the inhibition of COMT by CX and GM. Our results suggest that YKS facilitates the DA supplemental effect of L-DOPA, and that COMT inhibition by CX and GM contributes, at least in part, to the effects of YKS.

Yokukansan and Yokukansankachimpihange Ameliorate Aggressive Behaviors in Rats with Cholinergic Degeneration in the Nucleus Basalis of Meynert

Yokukansan (YKS) and yokukansankachimpihange (YKSCH) are traditional Japanese Kampo medicines. The latter comprises YKS along with the medicinal herbs Citrus unshiu peel and Pinellia tuber. Both of these Kampo medicines are indicated for the treatment of night crying and irritability in children and for neurosis and insomnia in adults. In recent clinical trials, YKS exhibited ameliorative effects on the behavioral and psychological symptoms of dementia, such as aggressiveness, excitement, and irritability. In the present study, we aimed to clarify the involvement of cholinergic degeneration in the nucleus basalis of Meynert (NBM) in the development of aggressiveness in rats. Subsequently, using this animal model, the effects of YKS and YKSCH on aggressiveness were compared and the mechanisms underlying these effects were investigated. L-Glutamic acid (Glu) was injected into the right NBM of rats to induce deterioration of cholinergic neurons. On day 8 after Glu injection, aggressive behaviors were evaluated using resident–intruder tests. After the evaluation, YKS or YKSCH was administered to rats with aggressive behaviors daily for 7 days. In some groups, the 5-HT1A receptor antagonist WAY-100635 was coadministered with YKS or YKSCH over the same period. In other groups, locomotor activity was measured on days 12–14 after Glu injection. On day 15, immunohistochemistry was then performed to examine choline acetyltransferase (ChAT) activities in the NBM. Aggressive behaviors had developed on day 8 after Glu injection and were maintained until day 15. YKS and YKSCH significantly ameliorated the aggressive behaviors. These suppressive effects were entirely abolished following coadministration of WAY-100635. Finally, the number of ChAT-positive cells in the right NBM was significantly reduced on day 15 after Glu injection, and treatment with YKS or YKSCH did not ameliorate these reduced cell numbers. Our results show that unilateral Glu injections into the NBM of rats leads to the development of aggressive behaviors, which is thought to reflect cholinergic degeneration. YKS and YKSCH treatments ameliorated Glu-induced aggressive behaviors, and these effects were suggested to be mediated by 5-HT1A receptor stimulation, but not by improvement of cholinergic degeneration.