Keiichi Samukawa

Facilitating action of asiaticoside at low doses on burn wound repair and its mechanism

Some reports published from 1967 to 1999 describe the use of ointments containing high doses (0.1 to 0.2%, w/w) C. asiataica herb extracts to enhance wound repair. Lower doses at which burn wound repair is enhanced by such topical applications have not been established yet. We found that the application of asiaticoside at low doses of 10(-8) to 10(-12)% (w/w) facilitated burn wound repair. To clarify the accelerating mechanisms of asiaticoside on burn wound repair, we examined the effects of asiaticoside on the levels of various cytokines produced at the site of the burn wound. The topical application of a low dose (10 pg, 1 ng, or 100 ng/wound area) of asiaticoside increased monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and interleukin (IL)-1beta levels in burn wound exudates. Asiaticoside (10 pg to 100 ng/ml) enhanced MCP-1 production in HaCaT cells, but it had no direct effect on VEGF production. Furthermore, asiaticoside (10 pg to 100 ng/ml) increased the IL-1beta production in THP-1 macrophages with MCP-1, but it had no effect on IL-1beta production without MCP-1 or with lipopolysaccharide (LPS). These findings suggest that the enhancement of burn wound healing by asiaticoside might be due to the promotion of angiogenesis during skin wound repair as a result of the stimulation of VEGF production caused by the increase in MCP-1 expression in keratinocytes and the increase in IL-1beta expression in macrophages induced cooperatively by asiaticoside plus MCP-1.

Prevention of Ischemic Neuronal Death by Intravenous Infusion of a Ginseng Saponin, Ginsenoside Rb1, That Upregulates Bcl-xL Expression

Almost all agents that exhibit neuroprotection when administered into the cerebral ventricles are ineffective or much less effective in rescuing damaged neurons when infused into the blood stream. Search for an intravenously infusible drug with a potent neuroprotective action is essential for the treatment of millions of patients suffering from acute brain diseases. Here, we report that postischemic intravenous infusion of a ginseng saponin, ginsenoside Rb1 (gRb1) (C54H92O23, molecular weight 1109.46) to stroke-prone spontaneously hypertensive rats with permanent occlusion of the middle cerebral artery distal to the striate branches significantly ameliorated ischemia-induced place navigation disability and caused an approximately 50% decrease in the volume of the cortical infarct lesion in comparison with vehicle-infused ischemic controls. In subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival, we showed that gRb1 stimulated the expression of the mitochondrion-associated antiapoptotic factor Bcl-xL in vitro and in vivo. Moreover, we revealed that a Stat5 responsive element in the bcl-x promoter became active in response to gRb1 treatment. Ginsenoside Rb1 appears to be a promising agent not only for the treatment of cerebral stroke, but also for the treatment of other diseases involving activation of mitochondrial cell death signaling.

Protective effect of ginsenosides Rg(2) and Rh(1) on oxidation-induced impairment of erythrocyte membrane properties.

The extract from Panax ginseng has been reported to improve the microcirculation in various organs. However, the mechanisms underlying this phenomenon are still poorly understood. In the present study, using the rheological properties of erythrocytes as an index, we have screened the components of Panax ginseng extract and identified Rg(2) and Rh(1) as the active ingredients. These two ginsenosides prevented the oxidative stress-induced elevation of erythrocyte suspension viscosity and the impairment of erythrocyte elongation in response to shear stress. Rg(2) and Rh(1) ginsenosides did not have antioxidant activity in an aqueous phase and did not inhibit the peroxidation of membrane lipids, either. However, they inhibited the oxidation-induced decrease of SH-groups in band 3 (anion exchanger-1), one of the important structural proteins of the erythrocyte membrane, but not in other structural proteins: bands 1 and 2 (spectrins), band 4.2 or band 5 (actin). These results suggest that ginsenosides Rg(2) and Rh(1) protect the rheological functions of erythrocytes against oxidative stress by preventing the oxidation of SH-groups in band 3 protein.

Intravenous Infusion of Ginsenoside Rb1 Ameliorates Compressive Spinal Cord Injury through Upregulation of Bcl-x L and VEGF

Background: Red ginseng root (Panax Ginseng C.A. Meyer) has been used clinically by many Asian people for thousands of years without any detrimental effects. One of the major components of red ginseng root is ginsenoside Rb1 (gRb1, C54H92O23, molecular weight 1109.46). Previously, we showed that intravenous infusion of gRb1 attenuated ischemic brain damage in rats through up-regulation of an anti-apoptotic factor, Bcl-xL. In the present study, we have investigated whether intravenous infusion of gRb1attenuates spinal cord injury or not. Methods: Adult male Wistar rats aged 12-14 weeks, weighing 250-300g, were used. Spinal cord injury (SCI) was induced by compressing the thoracic cord (Th12) with a 20 g weight for 20 minutes. Thirty minutes later, a total of 60µl gRb1 solution (12 or 60µg/60µl in saline) or the same volume of vehicle (saline) was infused once into the left femoral vein. Subsequently, continuous intravenous administration of gRb1 was performed for 7 days. The locomotor function was assessed using the Basso-BeattieBresnahan (BBB) score and the open field test at 1, 3 or 7days after SCI. The animals were killed at 7 days after SCI and the SCI lesions were assessed histologically. Results: Post-traumatic intravenous infusion of gRb1 (12µg/day or 60µg/day) ameliorated BBB score, rearing activity and neuronal density in the anterior horn of the damaged spinal cord at 7 days after SCI. Subsequent studies that focused on gRb1-induced expression of gene products responsible for neuronal death or survival revealed that gRb1 upregulated the expression of not only Bcl-x L , but also a potent angiogenic and neurotrophic factor, vascular endothelial growth factor (VEGF) at 7 days after SCI. Conclusion: Ginsenoside Rb1 (gRb1), with putative anti-apoptotic, angiogenic and neurotrophic actions, is a candidate agent for the treatment of patients with acute SCI.

Oral Administration of Red Ginseng Extract Promotes Neurorestoration after Compressive Spinal Cord Injury in Rats

Red ginseng and its active ingredients have been shown to decrease neuron death after brain ischemia in experimental animals. However, little is known about the effects of orally administered ginseng extract on spinal cord injury. We orally gave red ginseng extract (RGE) to rats with compressed spinal cord injury (SCI). Open-field locomotor scores were measured as indices of motor function. Histopathological changes and cytokine expressions in situ after SCI were evaluated. Compared to vehicle treatment, RGE treatment (350 mg/kg/day) significantly improved locomotor score up to levels close to those pre-SCI, prevented neuron loss, and facilitated the restoration of white matter in the spinal cord at 14 days after SCI. Treatment with RGE caused less aggregation of Iba-1-positive microglia in grey and white matter at 7 days after SCI, upregulated the expression levels of VEGF and Bcl-xL, and reduced IL-1β and TNFα expressions in the spinal cord at 7 and 14 days after SCI. We concluded that oral administration of RGE facilitates almost complete functional recovery from motor and behavioral abnormalities in rats with SCI and prevents neuron death in situ, possibly through inhibition of inflammation and upregulation of neuroprotective factors in the injured spinal cord.