Clinical significance of redox effects of Kampo formulae, a traditional Japanese herbal medicine: comprehensive estimation of multiple antioxidative activities.

To clarify the clinical significance of the redox-controlling effects of Kampo, a traditional Japanese herbal medicine, we determined the scavenging activities of various reactive oxygen species in clinically used Kampo formulae using an electron spin resonance-based technique. Formulae containing Rhei Rhizoma (i.e., mashiningan and daiobotanpito) showed high scavenging activity against the alkoxyl radical, and crude extract quantity was significantly correlated with scavenging activity. Hydroxyl radical scavenging activity was positively correlated with the quantity of Zingiberis Rhizoma. Strong hydroxyl radical scavenging activity was also found in formulae containing both Bupleuri Radix and Scutellariae Radix, a widely used anti-inflammatory combination. Formulae containing a clinically common combination of Scutellariae Radix, Coptidis Rhizoma, and Phellodendri Cortex induced high superoxide scavenging activity. Singlet oxygen scavenging activity was high in formulae containing Bupleuri Radix and Glycyrrhizae Radix. In contrast, formulae containing Rehmanniae Radix showed generally low reactive oxygen species scavenging activities, and the quantity of Rehmanniae Radix was negatively correlated with hydroxyl radical and singlet oxygen scavenging activities. These results indicate that the antioxidative effects of Kampo formulae are not uniform but complexly varied against multiple reactive oxygen species. Some formulae have almost no antioxidant effects but may act as pro-oxidants.

Effect of Keishibukuryogan, a Japanese Traditional Kampo Prescription, on Improvement of Microcirculation and Oketsu and Induction of Endothelial Nitric Oxide: A Live Imaging Study.

Oketsu is a characteristic condition that plays an important role in Kampo, Japanese traditional medicine, and includes multiple aspects of hemodynamic disorders. This study aims to clarify the microcirculation of Oketsu and the pharmacological effect of Keishibukuryogan, an anti-Oketsu Kampo prescription, using live imaging techniques. Oral administration of Keishibukuryogan induced significant vasodilation of murine subcutaneous arterioles compared to the preadministration level. This vasodilatation peaked 60 min after administration and persisted for 90 min. The blood velocity in the subcutaneous capillary was also increased by Keishibukuryogan in generally the same manner. In rat mesenteric arterioles, Keishibukuryogan administration improved microhemodynamic parameters, including the resolution of erythrocyte congestion and the cell-free layer, which are representative of Oketsu pathology. Live imaging revealed an increase of diaminofluorescein-2 diacetate fluorescence, a nitric oxide (NO) specific reagent, in the arterial endothelium following Keishibukuryogan administration. This fluorescence was most remarkable at vascular bifurcations but was present throughout the mesenteric arterioles. This study demonstrates the successful imaging of Oketsu pathology with respect to microcirculation and the anti-Oketsu effects of Keishibukuryogan, namely, vasodilation of arterioles, increased blood velocity, and resolution of erythrocyte congestion. The anti-Oketsu effect of Keishibukuryogan is related to endothelial NO production.

In vivo imaging of renal redox status during azelnidipine treatment.

The effect of the calcium channel blocker azelnidipine on the redox status of a murine hypertension model was analyzed and imaged using in vivo low frequency electron paramagnetic resonance (EPR). A murine two kidney-one clip (2K1C) hypertension model was produced by a clipping of the right renal artery. The resulting hypertensive mice were treated with low-dose azelnidipine (1 mg/kg/d), with high-dose azelnidipine (3 mg/kg/d) or without azelnidipine (HT group). An EPR system equipped with a loop-gap resonator and an imaging system was employed. Redox status was evaluated as organ reducing activity measured by means of the decay rate (half-lives) of the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL). Four weeks after clipping the mice demonstrated hypertension as expected. After the additional 2 weeks of azelnidipine treatments, the Carbamoyl-PROXYL half-lives of the Low and High azelnidipine groups measured in the upper abdominal area were significantly shorter than those of the HT group, suggesting improvements in the reducing activity. The blood pressures of the three groups showed no significant differences at this time, and there was no correlation between the renal reducing activity and either blood pressure or serum creatinine values. EPR imaging studies revealed that the improvement in abdominal reducing activity was mainly recognized in the kidney but not in the liver. These results indicate that azelnidipine ameliorates renal redox status through an improvement in reducing activity independent of blood pressure control.

Glycyrrhizae Radix attenuates peroxynitrite-induced renal oxidative damage through inhibition of protein nitration.

We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO- generation and NO production, were shown using a rat in vivo ONOO(-)-generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO- through scavenging ONOO- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO-.