加瀬 義夫

Medications or dietary components can affect both the host and the host's gut microbiota. Changes in the microbiota may influence medication efficacy and interactions. Daikenchuto (TU-100), a herbal medication, comprised of ginger, ginseng, and Japanese pepper, is widely used in Japanese traditional Kampo medicine for intestinal motility and postoperative paralytic ileus. We previously showed in mice that consumption of TU-100 for 4 weeks changed the gut microbiota and increased bioavailability of bacterial ginsenoside metabolites. Since TU-100 is prescribed in humans for months to years, we examined the time- and sex-dependent effects of TU-100 on mouse gut microbiota. Oral administration of 1.5% TU-100 for 24 weeks caused more pronounced changes in gut microbiota in female than in male mice. Changes in both sexes largely reverted to baseline upon TU-100 withdrawal. Effects were time and dose dependent. The microbial profiles reverted to baseline within 4 weeks after withdrawal of 0.75% TU-100 but were sustained after withdrawal of 3% TU-100. In summary, dietary TU-100 changed mouse microbiota in a time-, sex-, and dose-dependent manner. These findings may be taken into consideration when determining optimizing dose for conditions of human health and disease with the consideration of differences in composition and response of the human intestinal microbiota.

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.

The traditional Japanese herbal medicine hangeshashinto (HST) has beneficial effects for the treatment of oral ulcerative mucositis (OUM) in cancer patients. However, the ingredient-based mechanism that underlies its pain-relieving activity remains unknown. In the present study, to clarify the analgesic mechanism of HST on OUM-induced pain, we investigated putative HST ingredients showing antagonistic effects on Na+ channels in vitro and in vivo. A screen of 21 major ingredients using automated patch clamp recordings in channel-expressing cells showed that [6]-gingerol and [6]-shogaol, two components of a Processed Ginger extract, considerably inhibited voltage-activated Na+ currents. These two ingredients inhibited the stimulant-induced release of substance P and action potential generation in cultured rat sensory neurons. A submucosal injection of a mixture of [6]-gingerol and [6]-shogaol increased the mechanical withdrawal threshold in healthy rats. In a rat OUM model, OUM-induced mechanical pain was alleviated 30 min after the swab application of HST despite the absence of anti-bacterial and antiinflammatory actions in the OUM area. A swab application of a mixture of [61-gingerol and [6]-shogaol induced sufficient analgesia of OUM-induced mechanical or spontaneous pain when co-applied with a Ginseng extract containing abundant saponin. The Ginseng extract demonstrated an acceleration of substance permeability into the oral ulcer tissue without an analgesic effect. These findings suggest that Na+ channel blockage by gingerol/shogaol plays an essential role in HST-associated analgesia of OUMinduced pain. This pharmacological mechanism provides scientific evidence supporting the use of this herbal medicine in patients suffering from OUM-induced pain. (c) 2017 Elsevier Ltd. All rights reserved.

Cancer cachexia (CC) is a multifactorial disease characterized by decreased food intake and loss of body weight due to reduced musculature with or without loss of fat mass. Patients with gastric cancer have a high incidence of cachexia. We previously established a novel CC rat model induced by human gastric cancer-derived 85As2 cells in order to examine the pathophysiology of CC and identify potential therapeutics. In patients with CC, anorexia is often observed, despite elevation of ghrelin, suggesting that ghrelin resistance may develop in these patients. In this study, we aimed to clarify the occurrence of ghrelin resistance in CC rats accompanied by anorexia and we investigated whether rikkunshito (RKT), a traditional Japanese Kampo medicine that potentiates ghrelin signaling, ameliorated CC-related anorexia through alleviation of ghrelin resistance. 85As2-tumor-bearing rats developed severe CC symptoms, including anorexia and loss of body weight/musculature, with the latter symptoms being greater in cachectic rats than in non-tumor-bearing or pair-fed rats. CC rats showed poor responses to intraperitoneal injection of ghrelin. In CC rats, plasma ghrelin levels were elevated and hypothalamic anorexigenic peptide mRNA levels were decreased, whereas hypothalamic growth hormone secretagogue receptor (GHS-R) mRNA was not affected. In vitro, RKT directly enhanced ghrelin-induced GHS-R activation. RKT administrated orally for 7 days partly alleviated the poor response to ghrelin and ameliorated anorexia without affecting the elevation of plasma ghrelin levels in CC rats. The expression of hypothalamic orexigenic neuropeptide Y mRNA but not hypothalamic GHS-R mRNA was increased by RKT. Thus, the 85As2 cell-induced CC rat model developed ghrelin resistance, possibly contributing to anorexia and body weight loss. The mechanism through which RKT ameliorated anorexia in the CC rat model may involve alleviation of ghrelin resistance by enhancement of ghrelin signaling. These findings suggest that RKT may be a promising agent for the treatment of CC.

Maoto, a traditional Japanese Kampo medicine, has been used to treat various respiratory diseases, including respiratory infections and influenza. Ephedrine (EPD), the main ingredient in maoto, is also clinically used to treat respiratory diseases. However, the pharmacokinetics and distribution of EPD in the lungs after the administration of maoto have not been demonstrated. This study aimed to determine the concentrations, distribution, and pharmacokinetics of EPD and its precursor methylephedrine (MEPD) in the lungs of rats orally administered maoto (1 and 4 g/kg). We used liquid chromatography-electrospray ionization-tandem mass spectrometry to measure the ingredient concentrations. Both ingredients were detected in maoto-treated lung homogenates. Next, we examined the distribution of both ingredients in lung sections by using matrix-assisted laser desorption/ionization-mass spectrometry imaging, a powerful tool for the visualization of the distribution of biological molecules. The mass spectrometry imaging analysis detected only EPD and provided the first visual demonstration that EPD is distributed in the alveoli, bronchi, and bronchioles in the lungs of rats orally administered maoto (4 g/kg, three times at 2-h intervals). These results suggest that the pharmacological efficacy of maoto for the amelioration of respiratory symptoms is related to the distribution of EPD in the lung.

Objective: Recent studies have demonstrated that mouthwash made with the traditional Japanese medicine hangeshashinto exhibits anti-inflammatory action and alleviates oral mucositis scores, including pain complaints, in patients undergoing chemoradiotherapy. However, no study has demonstrated the mechanism underlying how hangeshashinto provides pain relief in oral ulcers. Design: The analgesic effects on pain-related behaviors following the topical application of hangeshashinto were evaluated in an oral ulcer rat model treated with acetic acid using recently developed methods. Indomethacin, the representative anti-inflammatory agent, was intraperitoneally administered. The tissue permeability of the oral mucosa was histologically evaluated after applying the fluorescent substance FluoroGold. Results: The topical application of hangeshashinto in ulcerative oral mucosa suppressed mechanical pain hypersensitivity over 60 min, without any effects on healthy mucosa. The same drug application also inhibited oral ulcer-induced spontaneous pain. Indomethacin administration failed to block the mechanical pain hypersensitivity, though it did largely block spontaneous pain. Topical anesthesia with lidocaine showed hyposensitivity to mechanical stimulation in healthy mucosa. In the ulcer regions in which the oral epithelial barrier was destroyed, deep parenchyma was stained with FluoroGold, in contrast to healthy oral mucosa, in which staining was limiting to the superficial site. Conclusions: Hangeshashinto leads to long-lasting analgesic effects, specifically in the ulcer region by destroying the epithelial barrier. Hangeshashinto alleviates oral ulcer-induced pain in inflammation dependent and/or independent manner. (C) 2016 Published by Elsevier Ltd.

Background Psychological stress has been shown to impair gastric accommodation (GA), but its mechanism has not been elucidated. This study was conducted to clarify the role of 5-HT2B receptors in a guinea pig model of stress-induced impairment of GA. Methods Gastric accommodation was evaluated by measuring the intrabag pressure in the proximal stomach after administration of a liquid meal. The guinea pigs were subjected to water-avoidance stress. The role of 5-HT2B receptors in impairment of GA was investigated by administering a 5-HT2B receptor agonist (BW723C86) or antagonist (SB215505), the traditional Japanese medicine rikkunshito (RKT), a muscarinic M-3 receptor antagonist (1,1-dimethyl-4-diphenylacetoxypiperidium iodide [4-DAMP]), or a nitric oxide synthase inhibitor (N-omega-nitro-L-arginine [L-NNA]). Key Results In normal animals, liquid meal-induced GA was inhibited by BW723C86, but was not affected by SB215505. The inhibition of GA by BW723C86 was reversed by co-administration of 4-DAMP. Compared to normal animals, GA in stressed animals was significantly inhibited. SB215505 and RKT significantly suppressed stress-induced impairment of GA. After meal administration, the level of cyclic guanosine monophosphate in gastric fundus tissue increased by approximately twofold in normal animals, but did not change in stressed animals. The inhibition of GA by L-NNA was suppressed by SB215505 or RKT. At a dose that did not affect GA in normal animals, BW723C86 exacerbated the impairment of GA in stressed animals. Conclusions and Inferences Stress-induced impairment of GA may be mediated by an increased responsiveness of 5-HT2B receptors, and activation of the 5-HT2B receptor signaling pathway may have an inhibitory effect on nitric oxide function.

This overview of herbal medicine use in Japan was designed to provide a review of the accumulating scientific evidence of the mechanism and clinical action of daikenchuto (DKT). Use of traditional Japanese medicines, including DKT, has a relatively “short” history of 500 years of clinical use. Only in the last 30 years has the Japanese government officially recognized herbal medicine as a valid form of treatment alongside the typical Western medicines. There has been a recent surge in scientifically robust data from basic and clinical studies for DKT, including placebo-controlled double-blind studies for various gastrointestinal disorders, and absorption, distribution, metabolism, and excretion studies have been conducted or are in the process of being conducted in both Japan and the USA. Clinical studies suggest that DKT is beneficial for postoperative ileus. Basic studies indicate that the effect of DKT is a composite of numerous actions mediated by multiple compounds supplied via multiple routes. In addition to known mechanisms of action via enteric/sensory nerve stimulation, novel mechanisms via the TRPA1 channel and two pore domain potassium channels have recently been elucidated. DKT compounds target these channels with and without absorption, both before and after metabolic activation by enteric flora, with different timings and possibly with synergism.

Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

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.

Keishibukuryogan is a traditional Japanese medicine widely administered to patients with menopausal symptoms. Because humans use it on a long-term basis, we believed that a carcinogenicity study was warranted. We orally administered keishibukuryogan (TJ-25) extract powder to 6-week-old Sprague-Dawley rats [Crl:CD(SD)], which were divided into four dosage groups-0 (water for injection), 100, 500 and 2,500 mg/kg/day for 24 months. We found that TJ-25 did not affect the survival rate of either sex. Furthermore, it did not affect the clinical condition of the rats, number of superficial tumors found by palpation, body weight, food consumption, hematology, or gross pathological findings. The severity of degeneration of muscle fiber in the femoral skeletal muscle increased slightly in males and females in the 2,500 mg/kg/day group, but TJ-25 did not increase the number of tumors found on histopathological examination. In our study, oral administration of TJ-25 extract powder in rats for 24 months was not associated with an increased incidence of tumors.

Oxaliplatin-induced peripheral neuropathy (OIPN) occurs at extraordinarily high frequency, but no effective treatment for this disorder has been established. Goshajinkigan (GJG), a traditional Japanese medicine known as Kampo, is known to reduce OIPN in both basic and clinical studies. However, its molecular mechanisms remain largely unknown. Here, we elucidate the mechanisms underlying the therapeutic effects of GJG against OIPN and the therapeutic benefits of combining GJG with bushi, a herbal medicine derived from the processed Aconiti tuber. Oxaliplatin (4 mg/kg) was injected into mice twice a week for up to 4 and 3 weeks, respectively. OIPN was assessed using pain behavioral tests, such as those testing cold hypersensitivity, thermal hyperalgesia, and mechanical allodynia, as well as a reduction of the current perception threshold (CPT). GJG (0.3 or 1 g/kg) and bushi (0.1 or 0.3 g/kg) were orally administered 5 times a week for 4 weeks. Behavioral analysis was performed 24 h after the final dose. Oxaliplatin induced cold hypersensitivity andmechanical allodynia but not thermal hyperalgesia and reduced CPT of A delta- and A beta-fibers but not C-fibers. All these effects were counteracted by GJG. Bushi, an ingredient of GJG that shows analgesic effect, reduced oxaliplatin-induced cold hypersensitivity but had no effect on oxaliplatin-induced mechanical allodynia. However, bushi significantly accentuated the effects of GJG when co-administered with GJG. GJG reduces OIPN by counteracting the sensitization of A delta- and A beta-fibers and shows analgesic effects against cold hypersensitivity and mechanical allodynia. These effects are potentiated by bushi. The combination of GJG with bushi has high potential for preventing OIPN.

Background: Hydrarthrosis, which is associated with knee pain and limited range of motion, decreases the quality of life (QOL) of patients with osteoarthritis (OA). The Kampo medicine boiogito is prescribed for the treatment of knee OA with hydrarthrosis; however, its precise mechanisms of action remain unknown. The purposes of this study were to assess the pharmacological effects of boiogito and its mechanisms of action on joint effusion in rats with surgically induced OA. Methods: A rat OA model was produced by transecting the anterior (cranial) cruciate ligament, medial collateral ligament, and medial meniscus in the right knee joints of 7-week-old female Wistar rats. The rats were given chow containing boiogito (1 or 2 %) or indomethacin (0.002 %) for 4 weeks after surgical transection. Levels of interleukin-1 beta (IL-1 beta) and hyaluronic acid (HA) were measured by enzyme-linked immunosorbent assay. Knee joint pain was assessed using an incapacitance tester. Osmotic water permeability in cultured rabbit synovial cells was assessed using stopped-flow analysis. Results: Increased synovial fluid volume and knee joint pain were observed in rats with surgically induced OA. In rats with OA, levels of IL-1 beta and HA in the articular cavity were higher but concentration of HA in synovial fluid was lower than in sham-operated rats, suggesting excessive synovial fluid secretion. Administration of boiogito improved hydrarthrosis, IL-1 beta, and HA concentrations and alleviated knee joint pain in rats with OA. Indomethacin reduced IL-1 beta and knee joint pain but failed to improve hydrarthrosis or HA concentration in rats with OA. Osmotic water permeability in synovial cells, which is related to the function of the water channel aquaporin, was decreased by treatment with boiogito. Conclusion: Boiogito ameliorates the increased knee joint effusion in rats with OA by suppressing pro-inflammatory cytokine IL-1 beta production in the articular cavity and regulating function of water transport in the synovium. The improvement of hydrarthrosis by boiogito results in the increased HA concentration in synovial fluid, thus reducing joint pain. Boiogito may be a clinically useful treatment of QOL in patients with OA with hydrarthrosis.

Shakuyakukanzoto (SKT), a traditional Japanese (Kampo) medicine, has been used by patients with muscle cramps and abdominal pains. In this trial, we analyzed plasma concentrations of active components after SKT was administered as a single oral dose of 2.5 or 5.0 g/day per person. The study was a randomized, open-label, two-arm, two-period, crossover trial conducted in healthy Japanese volunteers. Albiflorin (ALB), paeoniflorin (PAE), glycycoumarin (GCM), isoliquiritigenin (ILG), glycyrrhetic acid (GA), and glycyrrhetic acid-3-O-monoglucuronide were targeted, and the plasma concentration of each component was measured using a liquid chromatography-tandem mass spectrometry method. The pharmacokinetic parameters were calculated, and the linearity was assessed. All targeted components were detected in the plasma after oral administration of SKT. ALB, PAE, GCM, and ILG were detected at an early stage. The linearity was observed for the maximum plasma concentration of GCM, ILG, and GA and for the area under the plasma concentration-time curve of GA. In this trial, we demonstrated for the first time in humans that these components were absorbed into the blood after oral administration of SKT. The results of this pharmacokinetic trial in humans are also important and useful for understanding the mechanism of action of SKT, verifying the active components predicted in basic research, and conducting pharmacokinetics and safety studies in the future. (c) 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3952-3959, 2015

Oxaliplatin, a widely used chemotherapeutic agent, induces peripheral neuropathy that manifests itself as two distinct phases: acute cold hyperesthesia and chronic peripheral hypoesthesia/dysesthesia. The latter is a serious dose-limiting side effect that can often lead to withdrawal of treatment. We have developed a rat model expressing both phases and used the model to investigate the action of goshajinkigan (GJG), a traditional Japanese herbal medicine, which was reported to ameliorate oxaliplatin-induced neuropathy in a placebo-controlled double-blind randomized phase II study. In this study, neuropathy was induced by injection of oxaliplatin twice weekly for 8 wks. The maximum level of cold hyperesthesia was observed at 4 wks with heat hypoesthesia developing later. Microscopy studies revealed atrophy of axons of myelinated sciatic nerve fibers in oxaliplatin-treated rats at 8 wks. Co-administration of GJG ameliorated both abnormal sensations as well as histological damage to the sciatic nerve. A pharmacokinetic study revealed numerous neuroprotective components of GJG that are rapidly absorbed into the blood. GJG and some of its components attenuated the generation of oxaliplatin-induced reactive oxygen species, which is a possible mechanism of oxaliplatin-induced neurotoxicity. The present study provides a useful animal model for oxaliplatin-induced neurotoxicity as well as a promising prophylactic agent.

Most orally administered polyphenols are metabolized, with very little absorbed as aglycones and/or unchanged forms. Metabolic and pharmacokinetic studies are therefore necessary to understand the pharmacological mechanisms of polyphenols. Jumihaidokuto (JHT), a traditional Japanese medicine, has been used for treatment of skin diseases including inflammatory acne. Because JHT contains various types of bioactive polyphenols, our aim was to clarify the metabolism and pharmacokinetics of the polyphenols in JHT and identify active metabolites contributing to its antidermatitis effects. Orally administered JHT inhibited the increase in ear thickness in rats induced by intradermal injection of Propionibacterium acnes. Quantification by LC-MS/MS indicated that JHT contains various types of flavonoids and is also rich in hydrolysable tannins, such as 1,2,3,4,6-penta-O-galloyl glucose. Pharmacokinetic and antioxidant analyses showed that some flavonoid conjugates, such as genistein 7-O-glucuronide and liquiritigenin 7-O-glucuronide, appeared in rat plasma and had an activity to inhibit hydrogen peroxide-dependent oxidation. Furthermore, 4-O-methylgallic acid, a metabolite of Gallic acid, appeared in rat plasma and inhibited the nitric oxide reaction. JHT has numerous polyphenols; it inhibited dermatitis probably via the antioxidant effect of its metabolites. Our study is beneficial for understanding in vivo actions of orally administered polyphenol drugs.

Herbal medicines have been used in Japan for more than 1500 years and traditional Japanese medicines (Kampo medicines) are now fully integrated into the modern healthcare system. In total, 148 Kampo formulae are officially approved as prescription drugs and covered by the national health insurance system in Japan. However, despite their long track record of clinical use, the multi-targeted, multi-component properties of Kampo medicines, which are fundamentally different from Western medicines, have made it difficult to create a suitable framework for conducting well-designed, large-scale clinical trials. In turn, this has led to misconceptions among western trained physicians concerning the paucity of scientific evidence for the beneficial effects of Kampo medicines. Fortunately, there has been a recent surge in scientifically robust data from basic and clinical studies for some of the Kampo medicines, e.g., daikenchuto (TU-100). Numerous basic and clinical studies on TU-100, including placebo-controlled double-blind studies for various gastrointestinal disorders, and absorption, distribution, metabolism and excretion (ADME) studies, have been conducted or are in the process of being conducted in both Japan and the USA. Clinical studies suggest that TU-100 is beneficial for postoperative complications, especially ileus and abdominal bloating. ADME and basic studies indicate that the effect of TU-100 is a composite of numerous actions mediated by multiple compounds supplied via multiple routes. In addition to known mechanisms of action via enteric/sensory nerve stimulation, novel mechanisms via the TRPA1 channel and two pore domain potassium channels have recently been elucidated. TU-100 compounds target these channels with and without absorption, both before and after metabolic activation by enteric flora, with different timings and possibly with synergism.

Shakuyakukanzoto (SKT) is a kampo medicine composed of equal proportions of Glycyrrhizae radix (G. radix) and Paeoniae radix (P. radix). A double-blind study reported that SKT significantly ameliorated painful muscle cramp in cirrhosis patients without the typical severe side effects of muscle weakness and central nervous system (CNS) depression. Previous basic studies reported that SKT and its active components induced relaxation by a direct action on skeletal muscle and that SKT did not depress CNS functions; however, why SKT has a lower incidence of muscle weakness remains unknown. In the present study, we investigated which components are absorbed into the blood of rats after a single oral administration of SKT to identify the active components of SKT. We also investigated the effects of SKT and its components on the twitch contraction induced by physiological Ca2+ release. Our study demonstrated that SKT and five G. radix isolates, which are responsible for the antispasmodic effect of SKT, did not inhibit the twitch contraction in contrast to dantrolene sodium, a direct-acting peripheral muscle relaxant, indicating that the mechanisms of muscle contraction of SKT and dantrolene in skeletal muscle differ. These findings suggest that SKT does not reduce the contractile force in skeletal muscle under physiological conditions, i.e., SKT may have a low risk of causing muscle weakness in clinical use. Considering that most muscle relaxants and anticonvulsants cause various harmful side effects such as weakness and CNS depression, SKT appears to have a benign safety profile.

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy- and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study was to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (center dot OH)-scavenging ability and the reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin-trapping method. The superoxide (O-2(center dot-))-scavenging ability was estimated by an EPR redox probing method. Water suspensions of powdered HST and of its seven constitutive crude drugs were tested. In addition, some of the main water-soluble ingredients of the crude drugs were also tested. HST was found to scavenge both center dot OH and O-2(center dot-). Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso) and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good center dot OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water-soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function in maintaining catalytic redox action. The antioxidative function of HST probably worked via a balance of scavenging ROS, reducing stable free radicals, and some minor oxidizing activities.

Ethnopharmacological relevance: Yokukansan, a traditional Japanese (Kampo) medicine, has recently been used to treat the behavioral and psychological symptoms of dementia (BPSD), including aggressiveness, excitability, and hallucination. The present study was designed to investigate the mechanisms underlying the ameliorative effects of yokukansan on BPSD using animals exhibiting hallucination-like behaviors. For this purpose, we initially examined whether chronic isolation stress increases the frequency of hallucination in response to a psychedelic drug. Using this animal model, we next examined the effects of yokukansan on drug-induced hallucination-like behaviors. Finally, we examined the density and mRNA levels of serotonin 2A (5-HT2A) receptors. Materials and methods: Male mice were subjected to isolation stress for six weeks. Yokukansan was incorporated into food pellets, and administered to the mice for six weeks. In some experiments, yokukansan and each of seven constituent herbs were administered orally to the mice for the last two weeks during the six-week period of isolation stress. A 5-HT2A receptor agonist, 2,5-dimethoxy-4-iodoamphetamine (DOI, 2.5 mg/kg), was injected into the mice, and head-twitch behaviors were quantified. The binding sites of 5-HT2A receptors on the plasma membrane of the prefrontal cortex (PFC) were assessed by a receptor-binding assay using tritium-labeled ketanserin, and the density and affinity were calculated from a Scatchard plot. The level of mRNAs was measured by PCR analyses. Results: Isolation stress enhanced the frequency of the DOI-induced head-twitch response, and yokukansan treatment by feeding significantly reduced this enhancement Isolation stress significantly increased the 5-HT2A receptor density in the PFC, and yokukansan treatment by feeding as well as administration significantly down-regulated this increase. Isolation stress and yokukansan did not affect the affinity. Among seven constituent herbs, Bupleurum Root, Uncaria Hook, Japanese Angelica Root, and Glycyrrhiza down-regulated the increase, but statistically not significant, in which their efficacies were over 50% relative to yokukansan. Neither isolation stress nor yokukansan affected mRNA levels of 5-HT2A receptors. Conclusion: Yokukansan attenuated drug-induced hallucination-like behaviors in isolated mice, which is suggested to be mediated by 5-HT2A receptor down-regulation in the PFC. This mechanism may underlie the ameliorative effects of yokukansan on hallucination. (c) 2015 Elsevier Ireland Ltd. All rights reserved.

Geissoschizine methyl ether (GM) is an indole alkaloid found in Uncaria hook, which is a galenical constituent of yokukansan, a traditional Japanese medicine. GM has been identified as the active component responsible for anti-aggressive effects. In this study, the metabolic profiling of GM in rat and human liver microsomes was investigated. Thirteen metabolites of GM were elucidated and identified using a high-performance liquid chromatography with tandem mass spectrometry method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, and chromatographic retention times. There were no differences in the metabolites between the rat and human liver microsomes. Among the 13 identified metabolites, there were two demethylation metabolites, one dehydrogenation metabolite, three methylation metabolites, three oxidation metabolites, two water-adduct metabolites, one di-demethylation metabolite, and one water-adduct metabolite followed by oxidation. The metabolic pathways of GM were proposed on the basis of this study. This study will be helpful in understanding the metabolic routes of GM and related Uncaria hook alkaloids, and provide useful information on the pharmacokinetics and pharmacodynamics. This is the first report that describes the separation and identification of GM metabolites in rat and human liver microsomes.

Oral mucositis (OM) in cancer patients induced by chemotherapy or radiotherapy has a significant impact on quality of life, and causes considerable morbidity. Oral microorganisms are likely to intensify the inflammatory process and aggravate the formation of ulcers. Hangeshashinto (HST), a Japanese kampo medicine, has been reported to be effective when used as a gargle for the treatment of OM. To clarify the effects of HST on oral microorganisms, we assessed its antimicrobial activity against 27 microbial species, including 19 oral bacteria and one fungus. HST extract inhibited the growth of Gram-negative bacteria, including Fusobacterium nucleatum, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, Prevotella melaninogenica, Tannerella forsythia, Treponema denticola, and Porphyromonas asaccharolytica, though inhibitory effects were less pronounced for Gram-positive bacteria and the fungal strain. We then investigated the effects of antibacterial activities on 15 purified ingredients of HST and determined that baicalein, berberine, coptisine, [6]-shogaol, and homogentisic acid actively inhibited the growth of these bacteria. These findings showed that HST inhibits the growth of specific Gram-negative periodontopathogenic bacteria, which are significant pathogens in OM, without disturbing the normal oral flora. Our data suggest that HST may be a useful treatment for OM in patients undergoing anticancer treatment.

Effects of keishibukuryogan (KBG) on platelet aggregation were investigated. To ensure the specificity of KBG, tokishakuyakusan (TSS) and kamisyoyosan (KSS), which are known to have platelet aggregation-inhibiting effects, and rikkunshito (RKT) and shakuyakukanzoto (SKT), which are considered to be devoid of such effects, were used for comparison. The platelet aggregation of each test drug was measured by the screen filtration pressure method using whole blood of guinea pigs and expressed as a collagen-induced pressure rate (%) or a collagen concentration required for 50% increase in the pressure rate (PATI value). KBG suppressed the collagen-induced whole blood pressure rate increase and increased the PATI value, like TSS and KSS. Neither RKT nor SKT showed these effects. The Moutan cortex and Cinnamomi cortex, the constituent crude drugs of KBG, showed KBG-like pressure rate suppression and PATI-increasing effects. Furthermore, paeonol, a representative component of Moutan cortex, and aspirin which is known to have platelet aggregation-inhibiting activity (COX-1 inhibitor) also showed similar effects. These results suggest that the platelet aggregation-inhibiting activity of the constituent crude drugs Moutan cortex and Cinnamomi cortex is involved in the improving effects of KBG on impaired microcirculation and that paeonol plays a role in these effects.

The traditional Japanese medicine yokukansan has an anxiolytic effect, which occurs after repeated administration. In this study, to investigate the underlying mechanisms, we examined the effects of repeated yokukansan administration on serotonin 1A (5-HT1A) receptor density and affinity and its expression at both mRNA and protein levels in the prefrontal cortex (PFC) of socially isolated mice. Moreover, we examined the effects of yokukansan on a 5-HT1A receptor-mediated behavioral response. Male mice were subjected to social isolation stress for 6 weeks and simultaneously treated with yokukansan. Thereafter, the density and affinity of 5-HT1A receptors were analyzed by a receptor-binding assay. Levels of 5-HT1A receptor protein and mRNA were also measured. Furthermore, (+)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide (8-OH-DPAT; a 5-HT1A receptor agonist) was injected intraperitoneally, and rearing behavior was examined. Social isolation stress alone did not affect 5-HT1A receptor density or affinity. However, yokukansan significantly increased receptor density and decreased affinity concomitant with unchanged protein and mRNA levels. Yokukansan also enhanced the 8-OH-DPAT-induced decrease in rearing behavior. These results suggest that yokukansan increases 5-HT1A receptors in the PFC of socially isolated mice and enhances their function, which might underlie its anxiolytic effects.

Ethnopharmacological relevance: Geissoschizine methyl ether (GM) is an indole alkaloid that is a component of Uncaria Hook, and has been identified as the active component responsible for the anti-aggressive effects of the Uncaria Hook-containing traditional Japanese medicine, yokukansan. Recently, GM was shown to reach the brain by crossing the blood-brain barrier in rats following the oral administration of yokukansan. This finding suggested that there may be specific binding sites for GM in the brain. Here we show evidence that tritium-labeled GM ([H-3]GM) binds specifically to several brain areas of rats. Materials and methods: Male rats were used. [H-3]GM was synthesized from a demethylated derivative of GM. Specific binding sites of [H-3]GM on brain sections were determined by quantitative autoradiography, and maximum binding densities (B-max) and dissociation constants (K-d) were calculated. Several chemical compounds were used to clarify the molecules that recognize [H-3]GM in the completion-binding assay. Emulsion microautoradiography was also performed to identify the cells that bind [H-3]GM. Results: Specific binding of [H-3]GM was observed in the frontal cortex, including the prefrontal cortical region (e.g., prelimbic cortex (PrL)), hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus (DR), and cerebellum. B-max ranged between 0.65 and 8.79 pmol/mg tissue, and K-d was between 35.0 and 232.6 nM. Specific binding with relatively high affinity (K-d less than 62 nM) was dense in the frontal cortical region, moderate in the DR, and sparse in the cerebellum. The specific binding of [H-3]GM in the PrL was significantly replaced by the serotonin 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), 5-HT2A receptor antagonist ketanserin, 5-HT2B receptor agonist BW 723C86, 5-HT2C receptor agonist R060-0175, adrenergic alpha(2A) receptor antagonist yohimbine, L-type Ca2+ channel blocker verapamil, and mu-opioid receptor antagonist naloxone. Similar results were obtained in the frontal cortex and DR, but not in the cerebellum. Microautoradiography revealed that [H-3]GM signals were distributed throughout the frontal cortex, which included neuron-like large cells. Conclusion: These results demonstrate that specific binding sites for GM exist in rat brain tissue, and suggest that the pharmacological actions of GM are mainly associated with 5-HT receptors in the frontal cortex and DR. These results provide an insight into the neuropharmacology of GM and GM-containing herbal medicines. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of the kampo medicine yokukansan on gene expression of the cystine/glutamate antiporter system Xc(-), which protects against glutamate-induced cytotoxicity, were examined in Pheochromocytoma cells (PC12 cells). Yokukansan inhibited glutamate-induced PC12 cell death. Similar cytoprotective effects were found in Uncaria hook. Experiments to clarify the active compounds revealed that geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook, had cytoprotective effects. These components enhanced gene expressions of system Xc(-) subunits xCT and 4F2hc, and also ameliorated the glutamate-induced decrease in glutathione levels. These results suggest that the cytoprotective effect of yokukansan may be attributed to geissoschizine methyl ether, hirsuteine, hirsutine, and procyanidin B1 in Uncaria hook.

Background While there are reports that the herbal medicine rikkunshito (RKT) relieves upper gastrointestinal disease symptoms, the effect of RKT on primary afferent neurons is unknown. Methods A model of reflux esophagitis (RE) was implemented using male Wistar rats aged 6-7weeks. Ten days after surgery, the total area of esophageal mucosal erosion sites was determined. Th8-10 dorsal root ganglia (DRG) were dissected out and the expression of substance P (SP), calcitonin gene-related peptide (CGRP), and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) was determined in DRG using immunohistochemistry. RKT (0.6%/WV) or omeprazole (OME) (10mg/kg) was administered for 10days beginning on the day after surgery. Voluntary movement was measured with an infrared sensor for 22h each day. Key Results RE rats showed esophageal mucosal erosion and significantly increased number of SP/CGRP- and p-ERK1/2-immunoreactive neurons in DRG. Treatment with OME improved the size of erosive lesions in the esophageal mucosa of RE rats, while RKT did not. Treatment with RKT or OME significantly reduced the expression of SP/CGRP and p-ERK1/2 in DRG, and significantly increased voluntary movement in RE rats. Conclusions & Inferences RKT inhibited the activation of ERK1/2 and decreased the expression of SP and CGRP in DRG of RE rats, which may be associated with the observed amelioration of voluntary movement.

The acute peripheral neuropathy induced by oxaliplatin treatment occurs very frequently and is aggravated by exposure to cold. Goshajinkigan (GJG), a traditional Japanese (kampo) medicine, was recently shown to be effective against oxaliplatin-induced acute neuropathy. However, because the effects of GJG and its mechanism in relation to those of its ingredients and its mechanism are not well understood, we examined the effects of GJG on acute neuropathy. Further, we investigated whether GJG affects the functions and gene expressions of transient receptor potential (TRP) channels using a rat model of oxaliplatin-induced neuropathy. Administration of oxaliplatin increased withdrawal responses from cold stimulation, and GJG or calcium gluconate/magnesium sulfate significantly inhibited the oxaliplatin-induced cold hypersensitivity. Application of menthol, a TRPA1/TRPM8 agonist, or allyl isothiocyanate (AITC), a selective TRPA1 agonist, to the hind paw of oxaliplatin-treated rats enhanced the nocifensive behaviors evoked by each agonist, whereas oxaliplatin had no significant effect on nocifensive behaviors evoked by capsaicin, a TRPV1 agonist. GJG treatment reduced menthol- or AITC-evoked withdrawal responses potentiated by oxaliplatin. Furthermore, GJG suppressed the increase of TRPA1 and TRPM8 mRNA expression induced by oxaliplatin in dorsal root ganglia. These findings suggest that GJG prevented oxaliplatin-induced acute peripheral neuropathy by suppressing functional alteration of TRP channels, especially TRPA1 and TRPM8.

Cancer cachexia (CC), a syndrome characterized by anorexia and body weight loss due to low fat-free mass levels, including reduced musculature, markedly worsens patient quality of life. Although stomach cancer patients have the highest incidence of cachexia, few experimental models for the study of stomach CC have been established. Herein, we developed stomach CC animal models using nude rats subcutaneously implanted with two novel cell lines, i.e., MKN45c185, established from the human stomach cancer cell line MKN-45, and 85As2, derived from peritoneal dissemination of orthotopically implanted MKN45c185 cells in mice. Both CC models showed marked weight loss, anorexia, reduced musculature and muscle strength, increased inflammatory markers, and low plasma albumin levels; however, CC developed earlier and was more severe in rats implanted with 85As2 than in those implanted with MKN45cl85. Moreover, human leukemia inhibitory factor (LIF), a known cachectic factor, and hypothalamic orexigenic peptide mRNA levels increased in the models, whereas hypothalamic anorexigenic peptide mRNA levels decreased. Surgical removal of the tumor not only abolished cachexia symptoms but also reduced plasma LIF levels to below detectable limits. Importantly, oral administration of rikkunshito, a traditional Japanese medicine, substantially ameliorated CC-related anorexia and body composition changes. In summary, our novel peritoneal dissemination-derived 85As2 rat model developed severe cachexia, possibly caused by LIF from cancer cells, that was ameliorated by rikkunshito. This model should provide a useful tool for further study into the mechanisms and treatment of stomach CC.

Purpose: The aim of the study reported here was to determine the effect of surgical menopause by bilateral salpingo-oophorectomy (BSO) on circulating levels of cytokines and chemokines related to the pathogenesis of atherosclerosis. Patients and methods: A total of 110 women were recruited for this study from the outpatient clinic of our facility. We divided the women into three groups: 1) women with a regular menstrual cycle, 2) women in whom less than 5 years had passed since their BSO, and 3) women in whom 5 years or more had passed since their BSO. Concentrations of nine cytokines and chemokines in serum were measured. Results: The serum monocyte chemoattractant protein-1 (MCP-1) level in women in whom less than 5 years had passed since their BSO was significantly higher than in women with a regular menstrual cycle (P&lt 0.05). There were significant differences in serum interleukin (IL)-7 among the three groups (P=0.035). MCP-1 showed a significant positive correlation (r=0.320, P=0.008) with follicle-stimulating hormone in women with a regular menstrual cycle and in women in whom less than 5 years had passed since their BSO. Conclusion: A hypoestrogenic state due to BSO induced changes in MCP-1 and IL-7 levels. MCP-1 level showed a significant increase in the early period after BSO, while IL-7 level showed a significant decrease in the late period after BSO. © 2014 Tani et al, This work is published by Dove Medical Press Limited.

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&gt 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. © 2013 John Wiley &amp Sons, Ltd.

Purpose: Abnormal proximal gastric relaxation is one of the causes of functional dyspepsia. The purpose of this study is to use a barostat in conscious dogs to determine the effects of rikkunshito, which is considered to have beneficial effects on functional dyspepsia, on the proximal stomach. Methods: Eight beagles were used. A gastrocutaneous fistula and force transducers were surgically implanted in the middle corpus and gastric antrum and duodenum, respectively. After a recovery period, a plastic bag was inserted through the gastrocutaneous fistula and the proximal stomach was distended using a barostat First, four dogs were used to investigate the pressure volume relation in the fasted and postprandial phases. Second, the stomachs of four different dogs were continuously distended at minimal distending pressure +2 mm Hg, and 5 min later were infused with warmed liquid rikkunshito (2 g/20 mL) or water through the gastrocutaneous fistula. Finally, changes in the proximal gastric volume and gastrointestinal motility were observed. Results: The proximal stomach was significantly more pliable in the postprandial phase than in the fasted phase. The proximal gastric volume increased immediately after liquid infusion under constant pressure in both phases and duodenal motility was accelerated. The effect of rikkunshito was significantly greater and lasted longer than that of water. No significant difference between the effects during the fasted or postprandial phase and no change in the gastric antrum motility were observed when rikkunshito was infused. Conclusion: These results indicate that rikkunshito accelerates duodenal motility and relaxes the proximal stomach. (C) 2013 Elsevier B.V. All rights reserved.

Objective: The aim of the present study was to clarify the changes in circulating cytokines and chemokines in women during the menopausal transition by using a detailed classification. Materials and methods: A total of 554 women were recruited for this study from the outpatient clinic of the Department of Obstetrics and Gynecology, Tokushima University Hospital. We divided the women into seven stages by menstrual regularity and FSH level: mid-reproductive stage, late reproductive stage, early menopausal transition, late menopausal transition, very early postmenopause, early postmenopause and late postmenopause. We measured serum concentrations of nine cytokines (IL-1β, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, MIP-1β and MCP-1). Results: Serum IL-8 concentrations in postmenopausal women were significantly (p=0.001) higher than those in women in the mid- or late reproductive stage and women in early or late menopausal transition. Serum MCP-1 levels in women in late menopausal transition and postmenopause were significantly (p&lt 0.001) higher than those in women in the mid- or late reproductive stage and women in early menopausal transition. MCP-1 level showed a significant positive correlation (r=0.215, p&lt 0.01) with FSH level in women in menopausal transition. Conclusion: By using a detailed classification of menopausal transition, patterns of changes in IL-8 and MCP-1 levels during the menopausal transition were found to be different. IL-8 level showed a high level after menopause, while MCP-1 level showed a high level in menopausal transition. MCP-1 may be sensitive to hormonal change and may be involved in the development of estrogen deficiency diseases. © 2013 Elsevier Ltd.

Ethnopharmacological relevance: Shakuyakukanzoto (SKT) composed of Glycyrrhizae radix (G. radix) and Paeoniae radix (P. radix) has been traditionally used in Japan, Korea and China as an antispasmodic drug for the treatment of skeletal muscle cramps and intestinal cramps. Aim of this study: To evaluate the antispasmodic activity of SKT and its two components, as well as to identify the key constituents of the components which mediate this effect in skeletal muscles in vivo. Materials and methods: An experimental cramp model was constructed to evaluate the effects of peripherally-acting muscle relaxants on electrically-induced cramps under physiological conditions. This was accomplished by surgically isolating the motor supply to the gastrocnemius muscle in an anesthetized rat and delivering electrical stimuli to an isolated tibial nerve to induce tetanic contractions. We first tested dantrolene, a well-known peripherally-acting relaxant, to determine the sensitivity and reliability of our experimental model. We then evaluated the effects of SKT, P. radix, G. radix, and the eight active constituents of G. radix against tetanic contractions. Results: We found that dantrolene (10 and 30 mg/kg, i.d.) rapidly and significantly inhibited tetanic contractions (P&lt 0.01) irrespective of dose. SKT (0.5, 1.0, and 2.0 g/kg, i.d.) and G. radix (0.5 and 1.0 g/kg, i.d.) also significantly inhibited tetanic contractions (P&lt 0.01) but in a dose-dependent manner owing to the actions of six of the eight active constituents in G. radix (liquiritin apioside, liquiritigenin, isoliquiritin apioside, isoliquiritigenin, glycycoumarin, and glycyrrhetinic acid, 20 μmol/kg, i.v.). These constituents, which include flavonoids, a triterpenoid, and a courmarin derivative, demonstrated temporal variations in their inhibitory activity. In contrast, P. radix (0.5 and 1.0 g/kg, i.d.) did not show a statistically significant antispasmodic effect in our study however, we previously found that it had a significant antinociceptive effect. Conclusions: Our findings show that SKT inhibits tetanic contractions in vivo and that G. radix is the main antispasmodic component due to the actions of its active constituents, thus supporting the traditional use of SKT. We further propose that SKT containing the antispasmodic G. radix and antinociceptive P. radix is a pharmaceutically elegant option for muscle cramps as treatment requires a two-pronged approach, i.e. inhibition of hyperexcitable skeletal tissues and modulation of the pain accompanying cramps. © 2012 Elsevier Ireland Ltd.

Effects of seven alkaloids, geissoschizine methyl ether (GM), hirsutine, hirsuteine, rhynchophylline, isorhynchophylline, corynoxeine and isocorynoxeine, in Uncaria hook, a constituent of the kampo medicine yokukansan, on serotonin7 (5-HT7) receptor were investigated using Chinese hamster ovary (CHO) cell membranes and human embryonic kidney 293 (HEK293) cells stably expressing the human recombinant 5-HT7 receptor. A competitive binding assay using CHO membranes showed that GM (IC50 = 0.034 μM) more strongly inhibited the binding of the radioligand [3H] LSD to 5-HT7 receptor than the other alkaloids, suggesting that GM is bound to 5-HT7 receptor. Agonistic/antagonistic effects of GM (1-50 μM) on the receptor were evaluated by measuring intracellular cAMP levels in HEK239 cells. GM (IC50 = 6.0 μM) inhibited 5-HT-induced cAMP production in a concentration-dependent manner, as well as the specific 5-HT7 receptor antagonist SB-269970 (0.1-1 μM). However, GM did not induce intracellular cAMP production as 5-HT did. These results suggest that GM has an antagonistic effect on 5-HT 7 receptor. © 2012 Springer Science+Business Media, LLC.

Effects of yokukansan (YKS) on vacuous chewing movement (VCM), which is an index for tardive dyskinesia, were investigated in haloperidol decanoate-treated rats. Haloperidol decanoate was injected to a thigh muscle once every four weeks for 18 weeks. The rats which exhibited VCM eight times or more in 3 min were selected on the 12th week, and examined. A significant increase in VCM on the 12th week continued until the 18th week. Oral administration of VMS (0.1 and 0.5 g/kg) once a day for three weeks (21 days) from the 12th week to 15th week ameliorated the haloperidol decanoate-induced increase in VCM in a dose-dependent manner. The significant ameliorative effect observed in 0.5 g/kg VMS-treated rats was abolished by stopping administration for three weeks from the 15th week to the 18th week. The extracellular glutamate concentration and glutamate transporter mRNA expression in the striatum were evaluated by microdialysis and real-time reverse-transcription polymerase chain reaction assays at the 15th week. The striatal glutamate level increased in haloperidol-treated rats, and the increase was inhibited by treatment with VMS. The striatal GLT-1 mRNA level showed a tendency to decrease in the haloperidol-treated rats. The GLT-1 mRNA level after treatment with YKS (0.5 g/kg) was greater than the control level. These results suggest the effect of VMS may be involved in the extracellular glutamate level and GLT-1 mRNA expression in the striatum. (C) 2012 Elsevier Inc. All rights reserved.

Objective: The purpose of this study was to determine (1) the influence of estrogen deficiency induced by gonadotropin-releasing hormone (GnRH) agonist administration on insulin sensitivity as well as hormones and factors related to insulin resistance and (2) the differences in the influence for these parameters by the degree of basal insulin sensitivity. Methods: Thirty-five women diagnosed with leiomyoma were enrolled in this study. Serum levels of fasting glucose, insulin, sex steroid hormones, sex hormone-binding globulin (SHBG), vascular inflammatory markers and cytokines before and at 6 months after commencement of GnRH agonist administration were examined. Results: In all women, levels of insulin, glucose and homeostasis model assessment of insulin resistance (HOMA-IR) were not significantly changed. However, in women who had a low HOMA-IR before treatment, levels of insulin, glucose and HOMA-IR showed significant increases and total testosterone level showed a significant decrease. In women who had a high HOMA-IR, levels of insulin, HOMA-IR and SHBG were significantly decreased and levels of highly sensitive C-reactive protein, soluble intercellular adhesion molecule-1, E-selectin and monocyte chemoattractant protein-1 were significantly increased. Conclusion: Change in insulin sensitivity caused by GnRH agonist administration for premenopausal women with leiomyoma differs depending on baseline insulin sensitivity before treatment. (C) 2012 Elsevier B.V. All rights reserved.

Yokukansan (YKS), a traditional Japanese medicine, is composed of seven kinds of dried herbs. It is widely prescribed in clinical situation for treating psychiatric disorders such as aggressiveness in patients with dementia. We previously demonstrated that YKS and Uncaria hook (UH), which is a constituent herb of YKS, had a partial agonistic effect to 5-HT1A receptors in vitro. However, it has still been unclear whether this in vitro effect is reflected in in vivo, and what the active ingredients are. The purpose of the present study is to find the active ingredient in YKS and to demonstrate the effect in in vivo. In the present study, we first studied the effect of YKS and UH on aggressiveness and sociality in socially isolated mice. YKS and UH ameliorated the isolation-induced increased aggressiveness and decreased sociality, and these ameliorative effects were counteracted by coadministration of 5-HT1A receptor antagonist WAY-100635, or disappeared by eliminating UH from YKS. These results suggest that the effect of YKS is mainly attributed to UH, and the active ingredient is contained in UH. To find the candidate ingredients, we examined competitive binding assay and [S-35] guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding assay of seven major alkaloids in UH using Chinese hamster ovary cells expressing 5-HT1A receptors artificially. Only geissoschizine methyl ether (GM) among seven alkaloids potently bound to 5-HT1A receptors and acted as a partial agonist. This in vitro result on GM was further demonstrated in the socially isolated mice. As did YKS and UH, GM ameliorated the isolation-induced increased aggressiveness and decreased sociality, and the effect was counteracted by coadministration of WAY-100635. These lines of results suggest that GM in UH is potent 5-HT1A receptor agonist and a candidate for pharmacological effect of YKS on aggressiveness and sociality in socially isolated mice. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

We report the establishment of a new model for measuring gastric tone and liquid meal-induced accommodation in conscious guinea pigs and the role played by transient receptor potential ankyrin 1 (TRPA1). An indwelling polyethylene bag was placed in proximal stomachs of 5-week-old male Hartley guinea pigs. Gastric tone was measured by distending the bag and recording changes in intrabag pressure at various volumes. Gastric accommodation was measured by administering liquid meals and recording intrabag pressure over time. N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) (a nitric-oxide synthase inhibitor), atropine sulfate (atropine) (a muscarinic receptor antagonist), allyl isothiocyanate (AITC) (a TRPA1 agonist), or theophylline-7-(N-4-isopropylphenyl) acetamide (HC-030031) (a selective TRPA1 antagonist) was administered 15 to 60 min before measurement. Gastric tone was increased by stepwise distension of the bag and was further significantly increased by L-NAME and significantly decreased by atropine. A liquid meal (15% w/v; 1.7 kcal) significantly decreased intrabag pressure 5 to 20 min after administration, indicating gastric accommodation; this was completely suppressed by L-NAME and further enhanced by atropine. AITC significantly increased gastric tone; this increase was decreased by HC-030031 and atropine. A combination of AITC and L-NAME significantly increased gastric tone compared with L-NAME alone. HC-030031 alone significantly decreased gastric tone. Liquid meal-induced gastric accommodation was significantly suppressed by pretreatment with AITC. We established a new model for measuring gastric tone and accommodation in conscious guinea pigs. TRPA1 activation suppresses gastric accommodation by increasing gastric tone through cholinergic neuronal pathways.

Yokukansan, one of the traditional Japanese herbal medicines, ameliorated neuropathic pain symptoms in patients. In this study, we investigated the effects of yokukansan on neuropathic pain in chronic constriction injury (CCI) model. Oral administration of yokukansan significantly inhibited mechanical and cold allodynia in the von Frey hair or acetone test, respectively. In comparison, amitriptyline, a tricyclic antidepressant, demonstrated moderate, but not significant, antiallodynic effects in the mechanical and cold tests. Yokukansan significantly inhibited the cerebrospinal fluid dialysate level of glutamate that had increased by the stimulation of brush or acetone. Glutamate transporter inhibitors, DL-threo-beta-hydroxy aspartate and dihydrokainate, decreased the yokukansan-induced antiallodynic actions in CCI rats. Our results suggest that yokukansan was confirmed to have antiallodynic effects in CCI rats, which are related to a blockade of glutamatergic neurotransmission via activation of glutamate transporters in the spinal cord.