A simultaneous determination of principal compounds in tokishakuyakusan by high-performance liquid chromatography with diode array detector.

We developed a simultaneous analysis method using high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD) for six principal compounds (atractylenolide III, alisol A, alisol B, paeoniflorin, ferulic acid and (Z)-ligustilide) in a traditional Japanese (Kampo) medicine, tokishakuyakusan (TSS). The HPLC separation was conducted on a reversed-phase TSK-gel ODS-80TS column (4.6 i.d. × 250 mm, 5 µm) at 40°C with a 0.1% phosphoric acid-acetonitrile gradient system. The DAD detection wavelength was set at 205, 232 and 330 nm. Calibration curves for the compounds showed linear regressions with correlation coefficients of >0.999. The intra- and inter-day precision (i.e., the relative standard deviation) were in the range of 0.50-1.55 and 0.70-1.80%, respectively. The average recovery yields of the compounds ranged from 98.3 to 103%. The present results will contribute to shorter analysis times with less organic solvent compared with the individual analysis of each compound for the evaluation of TSS. The application of the established method to TSS will also provide helpful information for the further pharmacological and clinical studies.

New insights into the mechanism of neurolathyrism: L-ß-ODAP triggers [Ca2+]i accumulation and cell death in primary motor neurons through transient receptor potential channels and metabotropic glutamate receptors.

Neurolathyrism is a motor neuron (MN) disease caused by ß-N-oxalyl-L-α,ß-diaminopropionic acid (L-ß-ODAP), an AMPA receptor agonist. L-ß-ODAP caused a prolonged rise of intracellular Ca(2+) ([Ca(2+)]i) in rat spinal cord MNs, and the [Ca(2+)]i accumulation was inversely proportional to the MN's life span. The [Ca(2+)]i rise induced by L-ß-ODAP or (S)-AMPA was antagonized completely by NBQX, an AMPA-receptor blocker. However, blocking the L-type Ca(2+) channel with nifedipine significantly lowered [Ca(2+)]i induced by (S)-AMPA, but not that by L-ß-ODAP. Tetrodotoxin completely extinguished the [Ca(2+)]i rise induced by (S)-AMPA or kainic acid, whereas that induced by L-ß-ODAP was only attenuated by 65.6±6% indicating the prominent involvement of voltage-independent Ca(2+) entry. The tetrodotoxin-resistant [Ca(2+)]i induced by L-ß-ODAP was blocked by 2-APB, Gd(3+), La(3+), 1-(ß-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF-96365) and flufenamic acid, which all are blockers of the transient receptor potential (TRP) channels. Blockers of group I metabotropic glutamate receptors (mGluR I), 7-(hydroxyiminocyclopropan[b]chromen-1α-carboxylate ethyl ester (CPCCPEt) and 2-methyl-6-(phenylethynyl)-pyridine (MPEP) also lowered the [Ca(2+)]i rise by L-ß-ODAP. MN cell death induced by L-ß-ODAP was prolonged significantly with SKF-96365 as well as NBQX. The results show the involvement of TRPs and mGluR I in L-ß-ODAP-induced MN toxicity through prolonged [Ca(2+)]i mobilization, a unique characteristic of this neurotoxin.

Extraction efficiency of shosaikoto (xiaochaihu tang) and investigation of the major constituents in the residual crude drugs.

Shosaikoto (Xiaochaihu Tang) is one of the frequently used traditional herbal medicines (Kampo medicines, Japan). To realize the effective use of precious crude drugs, we investigated the major constituents in the residual crude drugs after decoction and aimed for extraction efficiency of shosaikoto with regard to the extract, tannin content, and major constituents. We found that the residual crude drugs had large amounts of compounds, especially saikosaponin b(2), which had a 78.3% yield compared to that in the first decoction. The extraction efficiency increased when decoction time and volume of water increased. Both increases had an additive effect on the yield of the extract and saikosaponin b(2) in particular. We also found that the size of crude drug pieces that are available in Japanese markets is suitable for decoction because of quick permeation of water. From our study, the second decoction may be a valuable contribution to medical treatment and of effective use of crude drugs. Moreover, time and volume of water should be increased when patients have trouble in preparing a decoction. Our study revealing the factors that influence the extraction efficiency of shosaikoto will be the basis for empirical evidence about decocting Kampo medicine.

Efficient preparation of Hangekobokuto (Banxia-Houpo-Tang) decoction by adding perilla herb before decoction is finished.

Hangekobokuto (banxia-houpo-tang) is a Kampo (traditional Japanese) medicine used mostly for anxiety disorder and consists of Pinellia Tuber, Poria Sclerotium, Magnolia Bark, Perilla Herb and Ginger. Perilla Herb contains an essential oil rich in perillaldehyde, which has a pleasant flavor, but this is easily lost due to heating. We therefore investigated how the major constituents of hangekobokuto, namely perillaldehyde, rosmarinic acid, magnolol and [6]-gingerol, varied with time during decoction and approached an optimal condition for decoction. Rosmarinic acid at 15 min after boiling, and magnolol and [6]-gingerol at 30 min were mostly extracted, while 0.09 mg of perillaldehyde remained at 15 min, but was not detected at 30 min. From these results, a decoction was prepared by adding Perilla Herb 1, 2, 5 and 10 min prior to the termination time of decoction at 30 min. When Perilla Herb was added 1 min beforehand, perillaldehyde was abundant (1.58 mg) and the amount of rosmarinic acid was already the same as that in the conventional decoction at 30 min, but the amount of total extract was inadequate. When Perilla Herb was added 5 min beforehand, perillaldehyde remained to some extent (0.61 mg) and rosmarinic acid was significantly increased compared with that in the conventional decoction at 30 min. From these results, we can conclude that the decoction should be prepared by boiling for not more than 30 min and, if possible, Perilla Herb should be added 5 min prior to the termination time. Considering the antidepressant-like activity of perillaldehyde and rosmarinic acid, the suggested method will not only achieve better treatment for anxiety, but also provide an effective use of crude drugs in the resource-limited environment.

Sulfur amino acids deficiency caused by grass pea diet plays an important role in the toxicity of L-ß-ODAP by increasing the oxidative stress: studies on a motor neuron cell line.

Neurolathyrism is a motor neuron disease caused by the overconsumption of grass pea (Lathyrus sativus L.) containing L-ß-ODAP. The precise mechanism to cause motor neuron degeneration has yet to be elucidated, but should agree with the epidemiological backgrounds. Considering the amino acid content of the legume, and the epidemiological link with prolonged unbalanced nutrition, the shortage of sulfur amino acids methionine and cysteine could affect the toxicity of L-ß-ODAP. We analyzed the effect of these amino acids in the media on the toxicity using primary motor neuron culture and a motor neuron cell line NSC-34. Deprivation of both methionine and cysteine exacerbated the toxicity of L-ß-ODAP by 66% compared to the complete medium. The glutathione content of these cells was greatly decreased in sulfur amino acid-deprived medium. L-ß-ODAP further lowered the content in the deprived media to be 32-44% of the controls compared to normal media being 62-74%. The increased motor neuron toxicity in this medium was neutralized by the addition of reduced glutathione ethyl ester or N-acetylcysteine suggesting the importance of the mitochondrial oxidative stress induced by L-ß-ODAP under sulfur amino acid-deficient conditions.

Hind-limb paraparesis in a rat model for neurolathyrism associated with apoptosis and an impaired vascular endothelial growth factor system in the spinal cord.

Neurolathyrism is a motor neuron disease characterized by lower limb paraparesis. It is associated with ingestion of a plant excitotoxin, beta-N-oxalyl-L-alphabeta-diaminopropionic acid (L-beta-ODAP), an agonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-type glutamatergic receptors. Previously, a limited model of neurolathyrism was reported for the rat. To improve upon the model, we stressed rat pups by separation from their mothers, followed by the subcutaneous L-beta-ODAP treatment, resulting in a 4.6-fold higher incidence (14.0-15.6%) of the paraparesis compared with the prior study. The number and size of motor neurons in these rats were decreased only in the lumbar and sacral cord segments, at approximately 13-36 weeks after treatment. Only lumbar and sacral spinal cord tissue revealed pathological insults typical of physical and ischemic spinal cord injury in the surviving motor neurons. In addition, extensive but transient hemorrhage occurred in the ventral spinal cord parenchyma of the rat, and numerous TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive cells were also observed. In parallel, vascular endothelial growth factor receptor (VEGFR)-2 (Flk-1) levels were significantly lowered in the lumbosacral spinal cord of the paraparetic rats compared with their controls, suggesting a failure of the VEGF system to protect neurons against L-beta-ODAP toxicity. We propose, based on these data, a novel pathological process of motor neuron death induced by peripheral L-beta-ODAP. For the first time, we present a model of the early molecular events that occur during chemically induced spinal cord injury, which can potentially be applied to other neurodegenerative disorders.

A new lignan from Balanophora abbreviata and inhibition of lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression.

Six lignans including a new lignan (1), beta-sitosterol glucopyranoside and phenylpropanoids were isolated from the whole plants of Balanophora abbreviata Bl. (Balanophoraceae). Their structures were determined by NMR, MS analysis and other spectroscopic methods. Lignans (1, 2 and 4) showed potent inhibitory activities on the lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells.

Partial involvement of group I metabotropic glutamate receptors in the neurotoxicity of 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-beta-ODAP).

Neurolathyrism is a human motoneuron disease caused by the overconsumption of grass pea (Lathyrus sativus) that contains a toxic non-protein amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (L-beta-ODAP). The preventive activities of various glutamatergic agents from acute neuronal death caused by L-beta-ODAP were studied using rat primary cortical neuron/glia culture. Nearly 80% of the rat primary cortical neurons were killed by 300 microM L-beta-ODAP within 24 h. Though antagonists acting on the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor prevented most of the toxicity, antagonists acting on group I metabotropic glutamatergic receptors (mGluRs), including (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and 2-methyl-6-(2-phenylethenyl)pyridine (SIB1893) partially and significantly prevented neuronal death due to L-beta-ODAP. These antagonists, within limited concentrations, did not have any inhibitory effects on the currents through AMPA receptors expressed in Xenopus oocytes. L-beta-ODAP itself did not induce the currents through group I mGluRs expressed in Xenopus oocytes. These results suggest that the neurotoxicity induced by L-beta-ODAP is partially mediated by the activation of group I mGluRs by an indirect mechanisms.

Toxicological considerations of Kampo medicines in clinical use.

Kampo (Japanese herbal) medicines, produced by combining multiple crude drugs, almost all of plant origin but with some of animal or mineral origin, contain great many substances. Since Kampo medicine results from the combination of many substances, their effect is a combination of the various interactions of the constituent substances. It has been demonstrated that several potential side effects such as allergic reactions, diarrhea and vomiting may be experienced when administering Kampo medicine. In addition, it has been reported that Kampo medicine may have antagonistic or synergistic interactions with western drugs or with some foods such as grapefruit juice. Among the 210 medicinal prescriptions used in present-day Japan, the clinical uses and the acute, chronic and mutagenic toxicity of some Kampo medicines are described. Studies of some potential interaction between Kampo medicines and western drugs are also considered.

Toxicological aspects of Kampo medicines in clinical use.

Among 210 medicinal prescriptions used in present-day Japan, the clinical uses and the acute, chronic and mutagenic toxicity study of 16 Kampo (Japanese herbal) medicines are summarized. These Kampo medicines are classified into two categories; eight prescriptions containing Bupleurum root (Bupleurum falcatum L.) such as Sho-saiko-to and Saiko-keishi-to, and eight prescriptions not containing Bupleurum root such as Juzen-taiho-to and Ninjin-yoei-to. Studies of some potential interaction between herbal medicine and western drugs are also described.

A new ent-clerodane diterpene from the aerial parts of Baccharis gaudichaudiana.

A new ent-clerodane diterpene, named bacchariol (1) was isolated from the aerial parts of Baccharis gaudichaudiana DC. (Compositae), together with known ent-clerodane diterpenes (2, 3), eight known flavonoids (4-11) and 3, 5-dicaffeoylquinic acid (12). Their structures were determined by spectroscopic analyses. Flavonoids (7, 8, 11) and 12 showed moderate scavenging activities toward 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals.

Neuroactive and other free amino acids in seed and young plants of Panax ginseng.

The seeds and one to three years old plants of Asian ginseng (Panax ginseng C.A. Meyer) were analyzed for their free amino acid contents. The neuro-excitatory beta-ODAP (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid), suggested to be the cause of the crippling neurolathyrism, was the major component in the seed extract (70% of the total free amino acids detected) and showed the highest concentration (0.43% by wt) compared to that in the different parts of young plants. beta-ODAP concentration was higher in the shoots as compared to roots and declined in older plants. The amount of beta-ODAP in the roots may be considered as an indirect measure of age and quality. Another neuro-active non-protein amino acid, GABA (gamma-aminobutyric acid), increased dramatically after germination and reached highest concentration in different parts of 3 year-old plants. Glutamine and arginine were the two major free proteinogenic amino acids in the ginseng plants and together they constituted over 50% of all the free amino acids detected in the root.

Ardisiphenols and other antioxidant principles from the fruits of Ardisia colorata.

Novel alkylphenols, ardisiphenols A-C (1-3) and a novel bergenin derivative, demethoxybergenin (10) were isolated from the fruits of Ardisia colorata (Myrsinaceae), together with known alkylresorcinols (4-6), embelin (7), myricetin (8), quercetin (9), bergenin (11), norbergenin (12), kaempferol (13), quercetin-3-O-beta-D-glucopyranoside (14) and gallic acid (15). Their structures were determined by NMR, MS(/MS) analyses and other spectroscopic methods. Ardisiphenols showed moderate scavenging activities toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and showed cytotoxicity against the murine breast cancer cell line, FM3A.

A new pentacyclic cucurbitane glucoside and a new triterpene from the fruits of Gymnopetalum integrifolium.

A new pentacyclic cucurbitane glucoside, named aoibaclyin (1) and a new triterpene (2) have been isolated from the EtOH extract of the fruits of Gymnopetalum integrifolium Kurz (Cucurbitaceae), together with three known compounds, bryoamaride (3), 25-O-acetylbryoamaride (4) and beta-sitosterol 3-O-beta-D-glucopyranoside (5). The structures of these compounds were elucidated by spectroscopic analyses.

Phenolic compounds from Gastrodia rhizome and relaxant effects of related compounds on isolated smooth muscle preparation.

Gastrol (1), together with 10 known phenolic compounds, has been isolated from the MeOH extract of the rhizomes of Gastrodia elata Blume (Orchidaceae), and their structures were elucidated by detailed spectral analyses including by 2D NMR spectroscopic analyses. The relaxant effects of these constituents on smooth muscle preparations isolated from guinea-pig ileum were also studied in order to reveal their characteristic pharmacological activities.