Pharmacology and toxicology of Bupleurum root-containing Kampo medicines in clinical use.

Kampo (Japanese traditional herbal) medicines have been produced by combining multiple crude drugs, almost all of plant origin but with some of animal or mineral origin, and contain a great many substances. Their effect is a combination of the various interactions of the constituent substances, whether they are enhancing, synergistic or suppressive. Kampo medicine has an overall effect that is different from the combined effects of individual crude drugs, and several side effects such as anorexia, slight fever and nausea have been reported in the treatment of certain disorders and disease states with Kampo medicines. Among 210 medical formulations used in Japan, some relevant information on the clinical uses, pharmacology and toxicology of six manufactured Kampo medical formulations, Shosaikoto, Daisaikoto, Saikokeishito, Hochuekkito, Saibokuto and Saireito, containing Bupleurum root are reviewed. Studies of some potential interactions between Kampo medicine and western drugs are also considered.

A rat model of neurolathyrism: repeated injection of L: -beta-ODAP induces the paraparesis of the hind legs.

Neurolathyrisim is a motor neuron disease characterized by spastic paraparesis in the hind legs, and is caused by grass pea, Lathyrus sativus, which contains the excitotoxic amino acid, 3-N-oxalyl-L: -2,3-diaminopropanoic acid (L: -beta-ODAP), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamatergic receptor agonist. In an attempt to make a useful model of this disease, the CNS distribution and toxicity of L: -beta-ODAP was studied in rat neonates after parenteral administration. L: -beta-ODAP was detected in the spinal cord as well as in the pons/medulla oblongata, though only small amounts in the latter. Repeated injection of L: -beta-ODAP resulted in rats with paraparesis of the legs, though at a low incidence rate of 0.032. These paralyzed rats displayed the severe atrophy of the ventral root of the lumbar cord as well as degenerations of motor neuron. The rats were useful models for the study of motor neuron degeneration in the spinal cord.

Wedelolactone suppresses LPS-induced caspase-11 expression by directly inhibiting the IKK complex.

Caspase-11 is a key regulator of proinflammatory cytokine IL-1beta maturation and pathological apoptosis. Caspase-11 is not expressed in most tissues under normal condition, but highly inducible upon pathological stimulation such as in the presence of lipopolysaccharide (LPS). Here, we describe the identification and characterization of wedelolactone, a natural compound that inhibits LPS-induced caspase-11 expression in cultured cells by inhibiting NF-kappaB-mediated transcription. We demonstrate that wedelolactone is an inhibitor of IKK, a kinase critical for activation of NF-kappaB by mediating phosphorylation and degradation of IkappaBalpha.

Ardisiphenols A-C, novel antioxidants from the fruits of Ardisia colorata.

Novel alk(en)ylphenols, named ardisiphenols A--C (1--3) were isolated from the fruits of Ardisia colorata, together with known alk(en)ylresorcinols (4--6). Their structures were determined by the NMR and MS/MS analyses. All compounds showed scavenging activities towards 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and cytotoxicities against murine breast cancer cell line, FM3A.

Sustained viral response is rarely achieved in patients with high viral load of HCV RNA by excessive interferon therapy.

Adequate dosing of interferon (IFN) and its cost-effectiveness for sustained virological response were evaluated in relation to viral load and subtype. Prospective analysis of IFN therapy on 326 patients with chronic hepatitis C free from cirrhosis was performed using 9 or 6 million unit (MU) of IFN for six months daily and/or three times a week. Sustained virological response was achieved in 50-94% of patients with < or =2 x 10(4) copies/ml (competitive RT-PCR) or <100 x 10(3) copies/ml (Amplicor monitor) of HCV RNA by 468-1206 MU of IFN, but response was only 0-25% of the patients with > or =2 x 10(5.5) copies/ml (competitive RT-PCR) or >200 x 10(3) copies/ml (Amplicor monitor), even with 468-1206 MU of IFN. A high sustained rate was demonstrated in patients with 100-200 x 10(3) copies/ml of HCV RNA by 901-1206 MU of IFN, in comparison to that with < or =900 MU of IFN. Multivariate analysis showed that IFN dose had a significant value for the efficacy of IFN therapy in patients presenting 100-200 x 10(3) copies/ml of HCV RNA. Cost efficacy analysis indicated that it cost approximately $10,000, $26,000, and $50,000-227,000 for one person-viral eradication in the patients with <100, 100-200, and >200 x 10(3) copies/ml, respectively. High-dose IFN is only cost effective in patients with intermediate viral loads, and IFN therapy could be recommended in patients with <200 x 10(3) copies/ml of HCV RNA.

Synthesis and pharmacological activity of O-(5-isoxazolyl)-L-serine.

A novel isoxazole derivative, O-(5-isoxazolyl)-L-serine (OIS, 1), was synthesized by a Mitsunobu reaction of isoxazolin-5-one (4) with N-Boc-L-serine tert-butyl ester (5) and subsequent deprotection of the coupling product. Its structure was elucidated by spectroscopic analyses. The pharmacological activity of 1 was also examined with cloned glutamate receptors and transporters using a Xenopus oocyte-expressing system showing substrate activity on an excitatory amino acid carrier 1 (EAAC 1) as a glutamate transporter.

Enzymatic formation of 2,3-diaminopropionic acid, the direct precursor of the neurotoxin beta-ODAP, in Lathyrus sativus.

The enzymatic breakdown of beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA) with formation of 2,3-diaminopropionic acid (DAPA), the direct precursor of the neurotoxin beta-ODAP in Lathyrus sativus, was confirmed in vitro. Some properties of the enzyme responsible for the biosynthesis of DAPA are described.

Clinical features of paradiverticulitis.

Colonic diverticula have been generally accepted as a source of massive hemorrhaging. Little is known, however, about fecal occult blood loss from colonic diverticula and diverticulosis. We retrospectively investigated the possibility of minor bleeding in 737 diverticula cases diagnosed from April 1989 to May 1994. Thirty-seven cases (5%) of diverticula are explained as the sources of positive occult blood testing ascertained clearly by colonoscopy. These divide into three types: (1) from intradiverticular bleeding (intradiverticulitis), (2) from peridiverticular bleeding (peridiverticulitis), and (3) from interdiverticular colonic mucosal erosion (interdiverticulitis). These findings account for the occult blood loss that we call paradiverticulitis. The two-year prospective study found 67 cases (11.3%) of paradiverticulitis in 595 diverticula cases. We concluded that paradiverticulitis is one of the causes of positive occult blood tests.

Effects on gastric circulation of treatment for portal hypertension in cirrhosis.

We evaluated the gastric circulatory effects of the type of treatment administered for portal hypertension. Of 14 patients with cirrhosis, seven received a transjugular intrahepatic portosystemic shunt (TIPS; group T) and seven received percutaneous transhepatic portographic embolization (PTPE; group P). Patients were evaluated over the course of one year. After treatment, portal venous pressure was significantly reduced from 39 +/- 6 cm H2O to 32 +/- 5 (P < 0.001) in group T and was significantly elevated from 29 +/- 10 to 33 +/- 8 (P < 0.05) in group P. The portal flow velocity (Vmean) was significantly higher in group T vs group P (P < 0.0001). The congestion index was significantly lower in group T than in group P (P < 0.0001). The gastric mucosal blood flow was increased in group T but was unchanged in group P. Esophageal varices showed some improvement in both groups, but the portal hypertensive gastropathy was improved only in group T. These findings help to explain the differing effects on the gastric circulation related to the type of treatment used for portal hypertension.

[Anti-dermaptophyte activity of phenolic compounds in "mokusaku-eki"].

"Mokusaku-eki," a kind of pyroligneous acid, is a dark brown color solution obtained from the charcoal burner of such Quercus spp. woods as a by-product. This is used as a folk medicine in water eczema. The n-hexane fraction of this solution contained several phenolic compounds such as 4-ethyl-2-methoxyphenol (3) and 2,6-dimethoxyphenol (4), which could not be found in the fresh woods. Among these compounds, 3 has the highest anti-dermaptophyte activity against Trichophyton mentagrophytes at minimum inhibitory concentration (MIC) 150 micrograms/ml.

Regulation of hepatic thrombopoietin production by portal hemodynamics in liver cirrhosis.

OBJECTIVE: This study was designed to clarify how thrombopoietin (TPO) functions in and, to some extent, causes thrombocytopenia complicating liver cirrhosis and portal hypertension. METHODS: Our study population consisted of 19 cirrhotic and six noncirrhotic patients who underwent percutaneous transhepatic portography (PTP) and hepatic venography. RESULTS: The platelet counts of the cirrhotic patients were significantly lower than those of the noncirrhotic patients (8.7 +/- 4.1 vs 17.4 +/- 7 x 10(4)/microl; p < 0.01). The flow direction in the splenic vein was confirmed by PTP. Ten hepatofugal and nine hepatopetal flow directions in the splenic vein were noted among the cirrhotics. The hepatofugal group showed lower portal venous pressure (20 +/- 10 vs 32 +/- 4 cm H2O; p < 0.01) than the hepatopetal group and had a higher incidence of hepatic encephalopathy (six of 10 vs zero of nine; p < 0.01). The hepatic vein-portal difference in TPO did not differ substantially between the cirrhotics and noncirrhotics (0.12 +/- 0.04 vs 0.24 +/- 0.07 fmol/ml). Comparisons of this value among the three groups showed the TPO difference to be lowest in the hepatofugal group (hepatofugal: 0.04 +/- 0.03, hepatopetal: 0.21 +/- 0.07, noncirrhotic: 0.24 +/- 0.07; p < 0.05). CONCLUSIONS: Our findings suggest that TPO production in the cirrhotic liver is regulated by the portal blood supply to the liver. Thus, portal hemodynamics may play a critical role in the development of thrombocytopenia.

Molecular cloning and characterization of the genes encoding two isoforms of cysteine synthase in the enteric protozoan parasite Entamoeba histolytica.

The enteric protozoan parasite Entamoeba histolytica was shown to possess cysteine synthase (CS) activity. The cDNA and genomic clones that encode two isoforms of the E. histolytica CS were isolated and characterized from a clonal strain of E. histolytica by genetic complementation of the cysteine-auxotrophic Escherichia coli NK3 with an E. histolytica cDNA library. The two types of the E. histolytica CS genes differed from each other by three nucleotides, two of which resulted in amino acid substitution. Deduced amino acid sequences of the E. histolytica CS, with a calculated molecular mass of 36721 Da and an isoelectric point of 6.39, exhibited 38-48% identity with CS of bacterial and plant origins. The absence of the amino-terminal transit peptide in the deduced protein sequences and the presence of the CS protein mainly in the supernatant fraction of the amoebic lysate after cellular fractionation suggested that the identified E. histolytica CS genes encoded cytosolic isoforms. Substrate specificity of the recombinant E. histolytica CS was similar to that of plant CS. Phylogenetic analysis indicates that the amoebic CS, first described in Protozoa, does not belong to any families of the CS superfamily, and represents a new family.

Beneficial effect of alpha-blocker on hemorheology in patients with essential hypertension.

To assess the effect of antihypertensive therapy on hemorheology in essential hypertension, blood viscosity and red blood cell deformability were examined in 45 patients with essential hypertension and 20 age-matched normotensive control subjects. Hypertensive patients were randomly assigned to monotherapy with five different antihypertensive drugs for 6 months and change of blood viscosity and red blood cell deformability were reexamined after the end of the monotherapy with each antihypertensive drug. Blood viscosity increased and red blood cell deformability decreased in hypertensive patients compared to normotensive control subjects. Monotherapy with each drug resulted in sufficient blood pressure control in all hypertensive patients. After the monotherapy with the alpha-blocker, terazosin, blood viscosity decreased significantly at shear rates from 22.5/sec to 450/sec, and red blood cell deformability, estimated by red blood cell filtration rate, increased by 15% (from 65 +/- 10 to 75 +/- 12 microL/sec, P < .05). The decrease in blood viscosity induced by alpha-blocker monotherapy may relate to an improvement of red blood cell deformability. It is possible that the treatment with alpha-blocker has a beneficial effect on the peripheral microcirculation due to an improvement of hemorheology in patients with essential hypertension.

[A long-term histological prognosis after IFN therapy for chronic hepatitis C].

In 72 chronic hepatitis C patients treated with IFN, liver biopsies were performed before and after IFN treatment, and histological changes were examined. In complete responders, the grading score improved significantly since 6-mo after IFN treatment, and the staging score improved significantly since 12-mo after IFN treatment. In long-term responders underwent a following liver biopsy 4-5 yr after IFN treatments; 5 had normal liver histology. In partial responders, both the grading and staging score did not improve significantly. But, in the cases who had sustained normalization of ALT levels after IFN treatment, both the grading and staging score improved after treatment. In non-responders, both the grading and staging score did not improve significantly.

Production of plant non-protein amino acids by recombinant enzymes of sequential biosynthetic reactions in bacteria.

We constructed the co-expression vector, pFK4, in which two cDNAs encoding serine acetyltransferase (SATase) and beta-(pyrazol-1-yl)-L-alanine/L-cysteine synthase (beta-PA/CSase) from Citrullus vulgaris (watermelon) were over-expressed under the transcriptional control of T7 promoter in Escherichia coli. Accumulation of both SATase and beta-PA/CSase in soluble extracts of E. coli was confirmed by immunoblotting. The high enzymatic activities of SATase and L-cysteine synthase (CSase) were detected in cell-free extracts of E. coli carrying pFK4. The activities of the formation of beta-PA and L-mimosine, plant non-protein amino acids, from O-acetyl-L-serine (OAS) and the precursor heterocyclic compounds, pyrazole and 3,4-dihydroxypyridine, were also found in the extracts. beta-PA was also produced in vivo from L-serine and pyrazole as precursors by E. coli cells transformed with pFK4. beta-PA was accumulated mainly in the extra-cellular culture medium. The pronounced accumulation of L-cysteine and L-methionine was observed in the cells transformed with pFK4. Additionally, we also constructed vectors which carried chimeric genes encoding fusion proteins of SATase and beta-PA/CSase. However, the fusion proteins tended to form insoluble inclusion bodies and thus to exhibit only weak enzymatic activities. The successful results of pFK4 shows the way to create a new sequential biosynthetic pathway of plant specific amino acids in bacterial cells by means of recombinant DNA technology.

Inhibitory activity of a naturally occurring heterocyclic beta-substituted alanine, beta-(isoxazolin-5-on-4-yl)-L-alanine, on the L-glutamate/L-aspartate transporter (GLAST) expressed in Xenopus oocytes.

Excitatory amino acid (EAA) transporters are of physiological importance in the regulation of the extracellular concentration of excitatory amino acids and the neuroexcitation in CNS. Among four identified transporters, the Na+-dependent high-affinity L-glutamate/L-aspartate transporter (GLAST) is highly expressed in glial cells. Here, we report a naturally occurring inhibitor of GLAST, derived from bovine retina, using the Xenopus oocyte expression system. Beta-(isoxazolin-5-on-4-yl)-L-alanine (TAN), an antifungal antibiotic, inhibited [14C]L-glutamate (L-Glu) transport into GLAST-expressing oocytes. TAN also served as a substrate for this transporter in voltage-clamp experiments measuring the current coupled to the EAA transport. The maximum current of TAN itself was approximately 1/3 of that of L-glutamate, and its apparent affinity was almost the same as L-Glu. In combination with L-Glu, TAN antagonized L-glutamate transport. In radioisotope experiments, the inhibitory potency of this compound against [14C]L-Glu uptake into oocytes was approximately 1/6 of that of L-(-)-threo-3-hydroxyaspartate (THA). The glucoside of TAN (TANG), occurring in seedlings of the garden pea, the lentil and some Lathyrus species, did not show any electrophysiological activity nor was it transported into oocytes. It is proposed that TAN is a novel type antagonist of natural origin on GLAST. By affecting such transport system, naturally occurring compounds may affect the regulation of the extracellular level of endogenous EAA.

Enzymatic synthesis of two isoxazolylalanine isomers by cysteine synthases in Lathyrus species.

Two isoxazolylalanine isomers, beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA, 1) and beta-(isoxazolin-5-on-4-yl)-L- alanine (TAN-950A, 2) were confirmed to be derived from O-acetyl-L-serine (OAS) and isoxazolin-5-one by cysteine synthases (CSases) with a different ratio in different plant parts. Some properties of this enzyme in the biosynthesis of both isomers are described.

Effects of ß-ODAP and its biosynthetic precursor on the electrophysiological activity of cloned glutamate receptors.

3-N-Oxalyl-l-2,3-diaminopropanoic acid (ß-ODAP) induces neurolathyrism, a motor neuron disease. To elucidate the pathogenic mechanism of this process, the action of ß-ODAP on the excitatory amino acid (EAA) receptor-mediated currents was examined using cloned EAA receptors expressed in Xenopus oocytes. On the voltage-clamp recordings of an AMPA receptor (α (1)α (2) heterooligomer), ß-ODAP was a strong agonist on this receptor, the potency being almost the same as l-glutamate. On the other hand, ß-ODAP had little effect on the glutamate-evoked currents through the expressed NMDA receptor (NR1(A)/NR2A), but showed a weak inhibitory effect on the glycine-modulatory site. ß-ODAP may cause the neurodegenerative disease, neurolathyrism, mainly through the excitotoxic interaction with AMPA receptors.

Purification and properties of a plant beta-D-glucuronidase form Scutellaria root.

beta-D-Glucuronidase (baicalinase, GUS [EC 3.2.1.31]) activity in the crude drug, Scutellaria root, was assayed in line with the quality control standards of Kampo (Japanese Herbal) medicines. GUS was purified to homogeneity in the purification steps including DEAE-Sepharose Fast Flow and chromatofocusing used PBETM94 and Polybuffer 74. These results suggest that the Scutellaria GUS is composed of 55kDa active subunits and that the isoelectric point of this enzyme is pH 5.4. Optimal catalytic activity was found at pH 4.7 in the pH range 3.6--6.2 in 50 mM Na-citrate buffer. The purified enzyme hydrolyzed baicalin and wogonin glucuronide, but did not hydrolyze glycyrrhizin or some beta-glucosides found in other crude drugs. GUS activity in several crude drugs is also described.

Interaction of some plant heterocyclic beta-substituted alanines with rat brain N-methyl-D-aspartate (NMDA) receptors.

The neuropharmacological actions of some plant heterocyclic beta-substituted alanines on rat brain N-methyl-D-aspartate (NMDA) receptors were studied. Of the compounds tested, 3-N-oxalyl-L-2,3-diaminopropanoic acid (beta-ODAP), the causal agent of human neurolathyrism, exhibited an inhibitory activity on the NMDA receptor binding assay at a relatively high concentration (IC50: 4.7 x 10(-5)M). The biochemical precursor of beta-ODAP, beta-(isoxazolin-5-on-2-yl)-L-alanine (BIA) was inactive in this assay. These results suggest that beta-ODAP, the neurotoxin of Lathyrus sativus, in addition to its excitatory action on alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptors, also has neurotoxic potential through its action on NMDA receptors.