Live-Imaging Analysis of Target Vessels and Nitric Oxide Production Associated with Gosha-Jinki-Gan and Keishi-Bukuryo-Gan: Two Herbal Preparations with Clinically Proven Blood Flow-Improving Effects but with Different Traditional Clinical Indicative Patterns.

Gosha-jinki-gan (GJG) and Keishi-bukuryo-gan (KBG) are Kampo traditional herbal prescriptions used for different clinical patterns (sho) that improve blood flow. The pharmacological basis of the therapeutic choice remains unclear, although the clinical reliance of this pattern-based therapy is widely proven. We aimed to investigate their effects on microcirculation and nitric oxide (NO) kinetics using a live-imaging system to provide evidence for this. Live-imaging was performed in murine subcutaneous vessels and rat mesentery. In the subcutaneous vessels, we analyzed the effects of both drugs on the vessel diameter, blood flow velocity, and volume in the arteries, arterioles, and capillaries. In the rat mesentery, we induced the "oketsu" blood stasis using a stack of thin vinylidene chloride films and examined the effect on NO production using a fluorescent diaminofluorescein-2 diacetate. Following dissolution in hot water, 300 mg/kg of both drugs were administered intragastrically via a transesophageal catheter. Live-imaging analysis of subcutaneous blood flow revealed the different effects of GJG and KBG on their target vessels and effect onset. GJG targeted the capillaries and progressively increased the blood flow velocity and rate at 30-120 min after administration. No vasodilation or increased blood flow in the arteries and arterioles occurred. In contrast, KBG increased the diameter of the arterioles and arteries at 30-90 min after administration, and increased blood flow velocity and rate in arteries and arterioles. In a model of oketsu blood stasis in the mesenteric arteries, KBG increased the NO production from the vascular endothelial cells with dilatation of the arteriolar diameter. GJG improved blood flow mainly in the capillaries. Endothelial NO production decreased after GJG administration. The empirical treatment choice between GJG and KBG is based on the difference in target vessels and NO action and provides a pharmacological basis for therapy based on traditional medicine.

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.

Therapeutic efficacy of the Qing Dai in patients with intractable ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that may become intractable when treated with conventional medications such as aminosalicylates, corticosteroids, and azathioprine. The herbal medicine Qing Dai has traditionally been used in Chinese medicine to treat UC patients, but there is a lack of published data on the efficacy of Qing Dai in UC treatment. We report several cases of patients with intractable UC who take Qing Dai in a retrospective observational study. Furthermore, we explore the mechanisms of action of Qing Dai. Nine patients with active UC who received conventional medications but wished to receive Qing Dai as an alternative medication were included in our analysis. The UC severity level was determined based on the clinical activity index (CAI). Additionally, 5 of the 9 patients were endoscopically evaluated according to the Matts grading system. Each patient received 2 g/d of Qing Dai orally and continued taking other medications for UC as prescribed. Electron spin resonance was applied to explore the mechanisms of action of Qing Dai. After 4 mo of treatment with Qing Dai, the CAI score decreased from 8.3 ± 2.4 to 2.4 ± 3.4 (mean ± SD; P < 0.001). Similarly, the endoscopic Matts grade decreased from 3.4 ± 0.5 to 2.2 ± 0.8 (P = 0.02). Six of 7 patients who were on prednisolone upon enrollment in the study were able to discontinue this corticosteroid. Electron spin resonance revealed that Qing Dai possesses strong hydroxyl radical scavenging activity. Qing Dai showed significant clinical and endoscopic efficacy in patients who failed to respond to conventional medications. Scavenging of hydroxyl radicals appears to be a potential mechanism through which Qing Dai acts, but the significance of the scavenging ability of Qing Dai with respect to the anti-inflammatory effect in UC patients warrants further investigation.

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.