Stimulation of cell-surface urokinase-type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin.

To investigate if neurotensin (NT) could induce activation of urokinase-type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl-NT (8-13) analogue, TJN-950, in which the C-terminal leucine is reduced to leucinol. TJN-950 inhibited the binding of 125I-NT to membranes of newborn rat brains and of COS-7 cells transfected with rat NT receptor cDNA, but at 10(4) higher doses than NT (8-13). However, TJN-950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN-950 induced neovascularization in the rat cornea in vivo. TJN-950 had no effect on expression of uPA, plasminogen activator inhibitor-1 or uPA receptor mRNA. The binding of 125I-TJN-950 to cell membranes was blocked by unlabeled uPA and TJN-950, but not the amino-terminal or 12-32 fragment of uPA. TJN-950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.

Suppressive effect of the herbal medicine Oren-gedoku-to on cyclooxygenase-2 activity and azoxymethane-induced aberrant crypt foci development in rats.

The present study is part of a program to obtain effective chemopreventive agents with low toxicity from medicinal herbs and traditional herbal medicines. We previously reported that Oren (Coptidis rhizoma) and Ogon (Scutellariae radix) inhibit azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation. In the present investigation, we found Sanshishi (Gardeniae fructus) and the traditional herbal medicine Oren-gedoku-to (OGT), composed of Ogon, Oren, Sanshishi and Obaku, also have preventive potential. Sanshishi and OGT decreased the numbers of ACF to 25.2 and 59.4% of the control value at 2% in the diet, respectively. Adverse effects, evidenced by body weight loss, were weaker with OGT than component herbs. To investigate their mechanisms of action, the influence on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) activities was studied. Both OGT and Sanshishi inhibited COX-2 but not COX-1, this presumably contributing to their suppressive effects on ACF development. The results suggest that OGT may be useful for colon cancer chemoprevention in terms of efficacy and toxicity.

Effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, on the survival of mice infected with influenza virus.

The antiviral effect of Hochu-ekki-to (TJ-41), a Japanese herbal medicine, was investigated using mice infected with influenza virus. TJ-41 was found to increase the survival rate, prolong the mean survival days, suppress viral growth in bronchoalveolar labage fluid (BALF) and inhibit the lung index (lung consolidation) on day 4 after infection in mice infected with influenza, after the agent had been administered orally once daily from day 7 to 2 before infection and from day 0 to 4 after infection. Administration of TJ-41 decreased the BALF concentrations of IL-1alpha, IL-6 and GM-CSF, but not TNF-alpha or interferon-gamma (IFN-gamma), on day 4 after infection. In addition, TJ-41 elevated the level of IFN-alpha in BALF on day 2 after infection. Yet, TJ-41 did not show any inhibitory effect on the growth of influenza virus in vitro. These results suggest that TJ-41 exerts its inhibitory effect on influenza virus infection via enhancement of the host immune responses in this experimental murine system.

In-vivo assessment of extrahepatic metabolism of paeoniflorin in rats: relevance to intestinal floral metabolism.

The extraction ratios of paeoniflorin in gut wall (EG), liver (EH) and lung (EL) were assessed by comparing AUCs after various routes of its administration to estimate the first-pass effects and the metabolism by intestinal flora. Pulmonary extraction ratio of paeniflorin was assessed by comparing AUCs calculated from venous and arterial plasma concentrations after its intravenous administration (0.5 mg kg-1). The mean pulmonary extraction ratio was estimated to be 0.06. The hepatic extraction ratio (EH was assessed by comparing AUCs after intraportal and intravenous administrations (0.5 and 5 mg kg-1). The plasma concentration profiles of paeoniflorin after intraportal administration were very close to those after intravenous administration, suggesting a negligible hepatic extraction ratio of paeoniflorin. The AUC value after intraperitoneal administration (0.5 mg kg-1) was greater than that after intraportal or intravenous administration. This finding suggests that paeoniflorin is not metabolized in the gut wall. The transference of paeoniflorin from the serosal side to the mucosal side was evaluated by the in-vitro everted sac method. The low intestinal permeability (19.4% at 60 min) was demonstrated by the comparison with phenobarbital (63.1% at 60 min). We conclude that paeoniflorin is not metabolize by gut wall, liver and lung, its poor absorption from the intestine results in extremely low bioavailability and the unabsorbed fraction of paeoniflorin is degraded by the intestinal flora.

Absorption and excretion of paeoniflorin in rats.

The absorption and excretion of paeoniflorin after intravenous and oral administration was studied in rats to evaluate the significance of paeoniflorin in the pharmacological action of Paeony root. The plasma concentration of paeoniflorin after intravenous administration at the doses of 0.5, 2.0 and 5.0 mg kg-1 rapidly decreased, simulated by a biexponential curve, with mean terminal half-lives of 11.0, 9.9 and 12.6 min, respectively. The Vdss values were 0.332, 0. 384 and 0.423 L kg-1 and the CLtot values were 26.1, 31.2 and 30.3 mL min-1 kg-1 at each dose. When given orally at the same doses, the absolute bioavailability values (F) determined by the AUC were 0.032, 0.033 and 0.038, respectively. The cumulative urinary and faecal excretions of paeoniflorin at the dose of 5 mg kg-1 after intravenous administration were 50.5 and 0.22% of the dose within 72 h, and 1.0 and 0.08% of the dose after oral administration within 48 h, respectively. Cumulative biliary excretion after intravenous or oral administration at a dose of 0.5 mg kg-1 was 6.9 and 1.3% of the dose within 24 h, respectively. The total CLR and CLB value after intravenous dosing was less than the CLtot value. These findings suggest that paeoniflorin is metabolized in other organs as well as in the liver. We conclude that paeoniflorin absorbed is excreted mainly in urine, it has a low bioavailability and the metabolites may be involved in the pharmacological action of Paeony root.

Bioavailability study of glycyrrhetic acid after oral administration of glycyrrhizin in rats; relevance to the intestinal bacterial hydrolysis.

To clarify the metabolic fate of glycyrrhizin when orally ingested, we investigated the bioavailability of glycyrrhetic acid, the aglycone of glycyrrhizin, after intravenous or oral administration of glycyrrhetic acid (5.7 mg kg-1, equimolar to glycyrrhizin) or glycyrrhizin (10 mg kg-1) at a therapeutic dose in rat. Plasma concentration of glycyrrhetic acid rapidly decreased after its intravenous administration, with AUC of 9200 +/- 1050 ng h mL-1 and MRT of 1.1 +/- 0.2 h. The AUC and MRT values after oral administration were 10600 +/- 1090 ng h mL-1 and 9.3 +/- 0.6 h, respectively. After oral administration of glycyrrhizin, the parent compound was not detectable in plasma at any time, but glycyrrhetic acid was detected at a considerable concentration with AUC of 11700 +/- 1580 ng h mL-1 and MRT of 19.9 +/- 1.3 h, while glycyrrhetic acid was not detected in plasma of germ-free rats at 12 h after oral administration of glycyrrhizin. The AUC value of glycyrrhetic acid after oral administration of glycyrrhizin was comparable with those after intravenous and oral administration of glycyrrhetic acid, indicating a complete biotransformation of glycyrrhizin to glycyrrhetic acid by intestinal bacteria and a complete absorption of the resulting glycyrrhetic acid from intestine. Plasma glycyrrhizin rapidly decreased and disappeared in 2 h after intravenous administration. AUC and MRT values were 2410 +/- 125 micrograms min mL-1 and 29.8 +/- 0.5 min, respectively. Plasma concentration of glycyrrhetic acid showed two peaks a small peak at 30 min and a large peak at 11.4 h, after intravenous administration of glycyrrhizin, with an AUC of 15400 +/- 2620 ng h L-1 and an MRT of 18.8 +/- 1.0 h. The plasma concentration profile of the latter large peak was similar to that of glycyrrhetic acid after oral administration of glycyrrhizin, which slowly appeared and declined. The difference of MRT values (19.9 and 9.3 h) for plasma glycyrrhetic acid after oral administration of glycyrrhizin and glycyrrhetic acid suggests the slow conversion of glycyrrhizin into glycyrrhetic acid in the intestine.

Effect of shosaikoto on plasma catecholamines.

Plasma adrenaline (AD) and noradrenaline (NA) were determined using high performance liquid chromatography (HPLC) equipped with an electrochemical detector. In an experiment using non-treated adrenalectomized rats, most plasma AD appeared to originate from the adrenal medulla, while plasma NA came from other sites. When Shosaikoto, a Japanese and Chinese traditional medicinal mixture, was orally administered to rats, plasma AD levels decreased in a significant manner 60 min after treatment, while plasma NA levels were not changed. Pretreatment with pentobarbital inhibited the Shosaikoto-induced decrease of plasma AD levels.

Effects of shosaikoto, an oriental herbal medicinal mixture, on restraint-stressed mice.

Restraint-induced stress produced an inhibition of humoral immune responses accompanied by a decrease of rectal temperature and motor activity. Diazepam and levamisole restored the decrease of humoral immune responses. Furthermore, diazepam restored the decrease of rectal temperature. Shosaikoto, an oriental herbal medicinal mixture, restored the stress-induced inhibition of humoral immune responses and rectal temperature.

Effects of certain herbal medicines on the biotransformation of arachidonic acid: a new pharmacological testing method using serum.

Two in vitro experimental systems were compared to evaluate the pharmacological effects of some herbal medicines on the biotransformation of arachidonic acid (AA) using the microsomal fraction of sheep vesicular glands as an enzyme source. The first system involved the addition of serum obtained from rats treated orally with the herbal medicines, while the second system involved the direct addition of herbal medicine extracts to the enzyme medium. Both systems were performed in parallel. Indomethacin, used as a reference drug, inhibited AA biotransformation in a dose-dependent manner in both experimental systems. The serum of rats orally pretreated for 1 h with Coptis japonica rhizome and Paeonia lactiflora root also inhibited AA biotransformation. Direct addition of hot water extracts of Rheum officinale rhizome, Scutellaria baicalensis root, Paeonia moutan bark and Zingiber officinale rhizome also inhibited AA biotransformation, while the extracts of Coptis japonica rhizome and Paeonia lactiflora root showed no effects.

Effect of shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide: a new pharmacological testing method.

Two in vitro experimental systems were compared to evaluate the pharmacological effects of Shosaikoto, a Japanese and Chinese traditional herbal medicinal mixture, on the mitogenic activity of lipopolysaccharide (LPS) using murine splenic cell culture. The first system involved the addition of serum obtained from mice treated orally with Shosaikoto while the second system involved the direct addition of Shosaikoto to the medium. Both systems were performed in parallel. Prednisolone decreased the mitogenic activity of LPS in a dose-dependent manner in both experimental systems. On the other hand, the serum of mice pretreated with Shosaikoto increased the mitogenic activity of LPS with constant cell viability, although the serum showed no mitogenic activity in the experiment without LPS. When Shosaikoto was added to the medium directly at more conventional doses, the mitogenic activity of LPS was elevated with an increase of cell viability. In the experimental system without LPS, Shosaikoto itself showed mitogenic activity. In other experiments, the mitogenic action of LPS was investigated using spleen cells of mice orally pretreated with Shosaikoto for 7 days. Mitogenic action of LPS was increased in the spleen cells of Shosaikoto-treated mice compared with the control. However, in the same experimental system without LPS, mitogenic action was not recognized in the spleen cells of Shosaikoto-treated mice.

Effect of shosaikoto, daisaikoto and sannoshashinto (traditional Japanese and Chinese medicines) on experimental hyperlipidemia in rats.

Effect of Sannoshashinto, Shosaikoto and Diasaikoto, Japanese and Chinese traditional medicinal mixtures (kampohozai), on cholesterol-induced hypercholesterolemia, aging-induced hyperlipidemia and cholesterol turnover were studied in rats. Sannoshashinto, Shosaikoto and Daisaikoto reduced the hypercholesterolemia induced by a high cholesterol diet and Sannoshashinto and Daisaikoto improved the atherogenetic index. Liver total cholesterol as increased by a high cholesterol diet was reduced by all three kampohozai. Furthermore, the increases of serum and liver triglyceride were also inhibited. In an experiment using untreated aging rats, both serum total cholesterol and serum triglyceride levels were increased. The aging-induced increases of serum total cholesterol were inhibited by Sannoshashinto and Daisaikoto and the increases of serum triglyceride were reduced by all three kampohozai. These drugs showed no effect on cholesterol biosynthesis in liver. Sannoshashinto, however, appeared to accelerate the disappearance of cholesterol from blood, while Daisaikoto inhibited the cholesterol absorption from the intestine.

Effects of five kampohozais on the mitogenic activity of lipopolysaccharide, concanavalin A, phorbol myristate acetate and phytohemagglutinin in vivo.

Mitogenic activity of lipopolysaccharide (LPS), concanavalin A (Con A), phorbol myristate acetate (PMA) and phytohemagglutinin (PHA) were investigated using spleen cells prepared from C57BL/6 mice orally pretreated with one of five kampohozai extracts (Shosaikoto, Daisaikoto, Hochuekkito, Juzendaihoto and Tokishakuyakusan). Shosaikoto and Daisaikoto elevated the mitogenic activity of LPS and reduced those of Con A. The mitogenic activities of PMA and PHA were elevated by low doses of Shosaikoto and Daisaikoto and suppressed by high doses. Juzendaihoto and Tokishakuyakusan showed no effect on the mitogenic activity of LPS and Con A, but increased those of PMA and PHA. Hochuekkito increased the mitogenic activity of LPS, Con A, PMA and PHA. In the absence of mitogens, these five kampohozais showed no mitogenic activity. These results indicate that the kampohozais used in this experiment appear to possess the immunomodulating or immunostimulating activities that might be expected from clinical experiences in Japan and China.

Antihepatotoxic actions of papyriogenins and papyriosides, triterpenoids of Tetrapanax papyriferum leaves.

Antihepatotoxic actions of the papyriogenins and papyriosides from Tetrapanax papyriferum leaves were examined utilizing carbon tetrachloride (CCl4)- and galactosamine (GalN)-induced cytotoxicity in primary cultured rat hepatocytes. Remarkable antihepatotoxic effects were observed with papyriogenin B, papyriogenin A, propapyriogenin A2, papyriogenin C, 11-dehydropropapyriogenin A2, 16-epi-saikogenin C and propapyriogenin A1 in the CCl4-induced cytotoxicity assay, and papyriogenin A, propapyriogenin A1 and propapyriogenin A2 were active in preventing GalN-induced cytotoxicity although these triterpenoids were rather cytotoxic at a higher dose in the latter assay. Structure-activity relationship is discussed.

Effects of shosaikoto, an oriental herbal medicinal mixture, on age-induced amnesia in rats.

The possibility of naturally occurring amnesia was examined in aging Fischer F254 rats. Animals 110 weeks old showed a loss of learning and memory in a passive avoidance responses (PAR) failure test. When 120 mg/kg/day of Shosaikoto extract was placed in the diet after 72 weeks of age to 110 weeks, it improved age-induced PAR failure in both the two-compartment avoidance box test and the step-down test. The responses of 110-week-old Shosaikoto-treated rats on both PAR failures were almost the same as those of non-treated 6-week-old rats. In these Shosaikoto-treated rats, dopamine was increased and norepinephrine and vanillyl mandelic acid were decreased in brain relative to those in non-treated aging rats.

Protective effect of hachimi-jio-gan, an oriental herbal medicinal mixture, against cerebral anoxia.

The protective effect of Hachimi-jio-gan (HJ) against cerebral anoxia was investigated with various experimental models in mice. Minimal effective dose of HJ which significantly prolonged the survival time was 0.5 g/kg, p.o. for normobaric hypoxia and 0.5 g/kg, p.o. for KCN- (4 mg/kg, i.v.) induced anoxia. HJ reduced the duration of coma induced by a sublethal dose of KCN (1.8 mg/kg, i.v.) in a dose-dependent manner. Furthermore HJ potentiated the anti-anoxic effect of physostigmine and the effect of HJ was diminished by the treatment with atropine.

Treatment of chronic liver injury in mice by oral administration of xiao-chai-hu-tang.

Oral administration of Xiao-Chai-Hu-Tang, the extract of a mixture of seven herbs, attenuated the hepatic fibrosis developed in mice after repeated doses of carbon tetrachloride. Its pre-administration reduced the derangement of liver function tests seen after a single dose of carbon tetrachloride as well as that seen after d-galactosamine intoxication. Xiao-Chai-Hu-Tang may be effective in the treatment of chronic liver injury through hepatocytoprotection.

Improving effects of the extracts from Eugenia uniflora on hyperglycemia and hypertriglyceridemia in mice.

EtOH (70%) extracts from the leaves of Eugenia uniflora were separated into six fractions with different polarity and molecular size, i.e. NP-1-NP-6. In an oral glucose tolerance test, NP-1 and 4 inhibited the increase in plasma glucose level. However, in an intraperitoneal glucose tolerance test, such an inhibitory effect was not seen. Thus, the effects of NP-1 and 4 were apparently due to the inhibition of glucose absorption from the intestine. In a sucrose tolerance test, all fractions inhibited the increase in plasma glucose level. In an oral corn oil tolerance test, NP-3 and 4 showed an inhibitory effect on the increase in plasma triglycerides level. On the other hand, NP-3, 4, 5 and 6 inhibited maltase and sucrase activities and all fractions except for NP-1 showed an inhibitory effect on lipase activity dose-dependently. The inhibition of the increase in plasma glucose level by NP-3, 4, 5 and 6 in the oral sucrose tolerance test and the inhibition of the increase in plasma triglycerides by NP-3 and 4 in the oral corn oil tolerance test were apparently due to the inhibition of the decomposition of carbohydrates and fats in the intestine, respectively.

Effect of Hachimi-jio-gan on scopolamine-induced memory impairment and on acetylcholine content in rat brain.

The effect of Hachimi-jio-gan (HJ) on scopolamine induced memory impairment was studied using a radial maze performance, the effect of HJ on the central cholinergic system as measured by acetylcholine (ACh) content, choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities was also examined. HJ (0.01-1.0 g/kg, p.o.) showed no influence on the radial maze performance. However, with the administration of scopolamine (0.5 mg/kg, i.p.), the number of the correct choices decreased and the number of the error choices increased. HJ (0.1 and 0.5 g/kg, p.o.) reduced this scopolamine-induced cognitive disturbance. The effect of HJ on ACh content and enzyme activities in the brain, frontal cortex, hippocampus and striatum was also investigated. In normal rats, HJ (0.1 and 0.5 g/kg, p.o. x 7 days) significantly increased ACh content in the frontal cortex, although it did not increased ACh content in the hippocampus. In scopolamine-treated rats, ACh content decreased in the brain regions examined. HJ (0.5 g/kg, p.o.) inhibited a decrease in ACh content in the frontal cortex, and with the same dosage of HJ increased CAT activity in the frontal cortex and AChE activity in the hippocampus. These results suggest that the behavioral effects of HJ may be related to its effect on the central cholinergic system.

Inhibitory effects of Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of atherosclerotic lesions in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

The inhibitory effects of the traditional herbal medicine Dai-saiko-to (Da-Chai-Hu-Tang) on the progression of the atherosclerotic lesions were studied using the spontaneous familial hypercholesterolemia (FH) model, Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. Changes in blood chemistry, pathology and low-density lipoprotein (LDL) oxidation were measured in a control group and a Dai-saiko-to-treated group. In the control group, the area of atheromatous plaques of the aorta progressed between week 12 (29.1%) and 26 (51.5%). This progression of atherosclerotic lesions did not happen in the Dai-saiko-to-treated group between week 12 (26%) and 26 (27.4%). Antioxidative effects on LDL were seen in the Dai-saiko-to-treated group in weeks 16 and 18. Dai-saiko-to did not improve the hypercholesterolemia in the KHC rabbits. These results suggest that Dai-saiko-to has inhibitory effects on the development of atheromatous plaque formation in spontaneous FH model rabbits. It is possible that the antioxidative effects of Dai-saiko-to on LDL led to the beneficial effects observed in this study.

Spectral analysis of body weight, food and water consumption and spontaneous motor activity in male Sprague-Dawley rats.

Spectral analyses by maximum entropy method were performed on the time-series data of body weight, food and water consumption and spontaneous motor activity in Sprague-Dawley rats. These parameters were recorded by a monitoring system collected every 30 min for 10 days. All the parameters had circadian (24 +/- 4 hr) and ultradian (less than 20 hr) rhythms. From the logarithmic transformation of spectral frequencies and their power spectral densities, the characteristics of the spectrum of body weight showed 1/f fluctuation and those of food and water consumption were white noises. The spectrum of the spontaneous motor activity showed complex characters. The range of frequencies lower than 0.1 Hz showed white noise while the range of those higher than 0.1 Hz were 1/f or 1/f2 fluctuations, especially in the range between 0.5 and 1.0 Hz (1-2 hours cycle). The auto-correlation coefficients of body weight did not declined suggesting that 24 hours cycle is only periodic phenomenon in body weight changes. Though the auto-correlation coefficients of food and water consumption also did not declined, they had not enough auto-correlations. This was consistent with the appearance of white noise in spectral analyses. The auto-correlation coefficients of spontaneous motor activity declined remarkably, and this suggests that there are various periodic changes in spontaneous motor activity including one to two hours cycles. These results suggest that the possibility of further extension of research is expected by using rhythmicity. It is also indicated that the time of measurement of body weight must be taken into consideration in experiments because of the presence of the circadian rhythmic changes.