Hepato-/reno-protective activity of Chinese prescription Kangen-karyu through inhibition of AGE formation and fibrosis-related protein expression in type 2 diabetes.

OBJECTIVES: This study was conducted to examine whether Kangen-karyu, a Chinese prescription, has an ameliorative effect on diabetes-induced alterations such as advanced glycation endproduct (AGE) formation or the fibrotic response in liver and kidney of type 2 diabetic db/db mice. METHODS: Kangen-karyu (100 or 200 mg/kg body weight/day, p.o.) was administered every day for 18 weeks to db/db mice, and its effect was compared with vehicle-treated db/db and m/m mice. KEY FINDINGS: The administration of Kangen-karyu decreased the elevated serum glucose concentration in db/db mice. The increased serum creatinine and urea nitrogen levels, which reflect renal dysfunction in db/db mice, were significantly lowered by Kangen-karyu administration. The db/db mice exhibited the up-regulation of AGEs and its receptor expression in liver and kidney; however, Kangen-karyu treatment significantly reduced expression except for the receptor. Moreover, the augmented expressions of fibrosis-related proteins, transforming growth factor (TGF)-ß1, fibronectin and collagen IV were down-regulated by Kangen-karyu administration. CONCLUSIONS: These results provide important evidence that Kangen-karyu exhibits a pleiotropic effect on AGE formation and fibrosis-related parameters, representing hepatoprotective and renoprotective effects against the development of diabetic complications in type 2 diabetic db/db mice.

Ameliorating effects of Kangen-karyu on neuronal damage in rats subjected to repeated cerebral ischemia.

We previously reported that 21-day (14-day pre-ischemic and 7-day post-ischemic) treatment with Kangen-karyu (KGKR) improved spatial memory impairment and hippocampal neuronal death induced by repeated cerebral ischemia (2 x 10-min, 1-h interval) in rats. In the present study, we examined the effect of single and 14-day pre-ischemic KGKR treatment on neuronal damage in the same repeated cerebral ischemia model. Additionally, to determine the mechanisms of neuroprotection by KGKR at glutamatergic neurons, we examined the effects of KGKR on glutamate release induced by repeated cerebral ischemia in vivo, and on cell damage induced by both glutamate and kainate in primary cultured hippocampal neurons in vitro. The 14-day pre-ischemic KGKR (300 mg/kg, oral administration (p.o.)) treatment reduced neuronal damage and astrocyte expression induced by repeated cerebral ischemia. No effect was observed after single pre-ischemic KGKR treatment. Both single and 14-day KGKR treatment decreased glutamate release in the hippocampal CA1 region in intact rats; however, neither pre-ischemic KGKR treatment altered glutamate release during cerebral ischemia. In vitro, KGKR (100-1000 microg/mL) dose-dependently suppressed hippocampal neuronal damage induced by both glutamate (100 microM) and kainate (1 mM). These data suggest neuroprotection with KGKR requires continuous pre-ischemic treatment, and that the mechanisms of protection may be involved in inhibiting the glutamatergic receptors of the post-synaptic neurons.

Neuroprotective effects of Kangen-karyu on spatial memory impairment in an 8-arm radial maze and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia in rats.

In the present study, we investigated the neuroprotective effects of Kangen-karyu (KGK) in a repeated cerebral ischemia model (2 x 10 min, 1-h interval). A 21-day pre- and post-ischemic treatment with KGK (10 - 300 mg/kg) and aspirin (5 mg/kg) improved the spatial memory impairment and neuronal death in the hippocampal CA1 region induced by repeated cerebral ischemia. However, a 7-day post-ischemic treatment with KGK did not attenuate the spatial memory impairment and neuronal death in this model. To determine the mechanism of action of KGK, we investigated the effects of a 14-day pre-ischemic treatment with KGK on cerebral blood flow in the hippocampal area of the repeated cerebral ischemia model using laser Doppler flowmetry. The 14-day pre-ischemic treatment with KGK increased the cerebral blood flow during reperfusion. These results suggest that a 21-day pre- and post-ischemic treatment with KGK can protect against brain damage caused by cerebral ischemia by increasing the cerebral blood flow in the hippocampal area.

Increase in the free radical scavenging activities of American ginseng by heat processing and its safety evaluation.

We previously reported the increase in free radical scavenging activities of Korean ginseng (KG, Panax ginseng C.A. Meyer) by heat processing. In the United States, American ginseng (AG, Panax quinquefolium L.) is a more commonly used herbal medicine than KG, but heat processing-induced chemical and activity changes of AG are not well known. Therefore, we compared the changes in ginsenosides, total phenolic contents, Maillard reaction product (MRP) levels, and several free radical scavenging activities of AG by heat processing. In addition, a short-term toxicity assessment in rats was also conducted for the identification of certain toxic effects of AG after heat processing. As a result, the ginsenosides were deglycosylated at carbon-20 and their total contents were lowered, but the total phenolic contents and MRP levels of AG were about 2.5 and 9.3 times increased, respectively, by heat processing. In addition, all free radical scavenging activities of AG were significantly increased by heat processing. Moreover, there were no toxic signs or decreases in renal and hepatic function parameters of rats administered heat-processed AG. Therefore, heat processing, as in KG, is a useful method to enhance the free radical scavenging activities of AG by the increases in total phenolic contents and MRP levels.

Protective effect of the Chinese prescription Kangen-karyu against high glucose-induced oxidative stress in LLC-PK1 cells.

We investigated the effects of Chinese prescription Kangen-karyu on high glucose-induced oxidative stress using LLC-PK(1) cells, renal tubular cells, which are the most vulnerable renal tissue to oxidative stress. High-concentration glucose (30mM) treatment induced LLC-PK(1) cell death, but Kangen-karyu, at a concentration of 5, 10 or 50 microg/ml, significantly inhibited high glucose-induced cytotoxicity. In addition, the intracellular reactive oxygen species level was increased by 30mM glucose treatment, but it was concentration-dependently inhibited by Kangen-karyu treatment. Moreover, 30mM glucose treatment induced high levels of superoxide anion, nitric oxide and peroxynitrite. However, Kangen-karyu treatment significantly reduced the radical overproduction induced by high glucose, suggesting Kangen-karyu has radical-scavenging activity that would protect against oxidative stress induced by high glucose. Kangen-karyu also reduced the overexpression of inducible nitric oxide synthase and cyclooxygenase-2 proteins induced by high glucose. Furthermore, treatment with Kangen-karyu, at a concentration of 50mug/ml, inhibited the nuclear translocation of nuclear factor-kappa B induced by 30mM glucose in LLC-PK(1) cells. These findings indicate that Kangen-karyu is a potential therapeutic agent that will reduce the damage caused by hyperglycemia-induced oxidative stress associated with diabetes.

The protective role of Chinese prescription Kangen-karyu extract on diet-induced hypercholesterolemia in rats.

This study was carried out to investigate the protective potential of Chinese prescription Kangen-karyu, comprising six crude drugs, on coronary heart disease which is the principal cause of morbidity and mortality worldwide. The diet-induced hypercholesterolemic rat model, which shows an elevation in low density lipoprotein (LDL) cholesterol and atherosclerosis, was employed. The control rats fed a diet of 1% cholesterol and 0.5% cholic acid showed the highest cholesterol levels in serum and feces relative to those fed a normal diet, however, the rats administered Kangen-karyu extract showed reductions in these levels without changes in liver cholesterol, indicating that the reduction of serum total cholesterol by Kangen-karyu extract probably arises from an increase in cholesterol excretion. Furthermore, the administration of Kangen-karyu extract significantly prevented the elevation of serum aspartate aminotransferase and alanine aminotransferase, known as marker enzymes of liver damage. The elevated serum levels of LDL cholesterol were lowered, however, the high density lipoprotein cholesterol level was significantly elevated by Kangen-karyu extract and these were dose-dependent decreases in the atherogenic index to 15.2, 8.8 and 7.5 at oral doses of 50, 100 and 200 mg from the 19.4 control value, respectively. In addition, Kangen-karyu extract inhibited LDL oxidation in a dose-dependent manner, and the elevated level of thiobarbituric acid-reactive substances in control rats showed a decline by the administration of Kangen-karyu extract. The present study suggests that Kangen-karyu could play a protective role against hypercholesterolemia through the regulation of cholesterol levels and inhibition of lipid peroxidation.

Effects of the Chinese prescription Kangen-karyu and its crude drug Tanjin on ageing process in rats.

The effects of the Chinese prescription Kangen-karyu and its crude drug Tanjin on the ageing process were investigated in rats. Diets supplemented with Kangen-karyu and Tanjin extracts decreased glycosylated protein levels in serum, a risk marker of ageing and ageing-related diseases. In addition, they inhibited the levels of thiobarbituric acid reactive substance in the serum and liver; Kangen-karyu in particular led to a strong decrease in hepatic mitochondrial thiobarbituric acid reactive substance. The decline in the reduced glutathione/oxidized glutathione ratio in the liver observed with ageing was ameliorated by Kangen-karyu and Tanjin, while these groups attenuated the increase in glutathione peroxidase activity of hepatic tissue against ageing. This suggests that Kangen-karyu and Tanjin regulate the glutathione redox cycle that maintains the cellular redox condition against age-related oxidative stress. Moreover, the overexpression of cytoplasmic cytochrome c observed with ageing was attenuated by Kangen-karyu and Tanjin. This provides new evidence that Kangen-karyu and Tanjin inhibit leakage of superoxide in mitochondria and attenuate cellular oxidative damage. Furthermore, Kangen-karyu and Tanjin would maintain mitochondrial function with ageing through the regulation of related protein expression such as bax and bcl-2 proteins. In addition, Kangen-karyu reduced the expression of nuclear factor kappa B; Kangen-karyu and Tanjin did not affect the expression of inhibitor kappa B. The present study demonstrated that Kangenkaryu prevented oxidative damage and mitochondrial dysfunction with ageing. Furthermore, Kangen-karyu showed a stronger protective effect against ageing by oxidative stress than Tanjin, probably through synergistic and/or additive effects.

The mechanisms underlying the anti-aging activity of the Chinese prescription Kangen-karyu in hydrogen peroxide-induced human fibroblasts.

Our previous study showed that Kangen-karyu extract protected against cellular senescence by reducing oxidative damage through the inhibition of reactive oxygen species generation and regulation of the antioxidative status. Although these findings suggest that Kangen-karyu could delay the aging process, the mechanisms responsible for protection against aging have rarely been elucidated. Therefore, this study was focussed on the mechanisms responsible for the anti-aging activity of Kangen-karyu extract using hydrogen peroxide (H(2)O(2))-induced human diploid fibroblasts, a well-established experimental model of cellular aging. Kangen-karyu extract exerted a protective effect against the morphological changes induced by H(2)O(2) treatment and inhibited senescence-associated beta-galactosidase activity. In addition, the beneficial effects of Kangen-karyu extract on cell viability and lifespan indicated that Kangen-karyu extract could delay the cellular aging process. The observation that Kangen-karyu extract prevented nuclear factor kappa B (NF-kappaB) translocation in response to oxidative stress suggested that Kangen-karyu exerted its anti-aging effect through NF-kappaB modulation and prevention of H(2)O(2)-induced overexpression of haem oxygenase-1 protein. Moreover, pretreatment with Kangen-karyu extract reduced overexpression of bax protein and prevented the mitochondrial membrane potential decline, suggesting that Kangen-karyu extract may protect mitochondria from mitochondrial oxidative stress and dysfunction. These findings indicate that Kangen-karyu is a promising potential anti-aging agent that may delay, or normalize, the aging process by virtue of its protective activity against oxidative stress-related conditions.

Antioxidative effects related to the potential anti-aging properties of the Chinese prescription Kangen-karyu and Carthami Flos in senescence-accelerated mice.

The popular oxidative stress theory predicts that enhancement of the antioxidative defense system to attenuate free radical-induced damage counteracts the aging process. We used senescence-accelerated mice (SAM) because SAM has been shown to suppress the antioxidative defense system and mitochondrial dysfunction induced by oxidative stress. We investigated the antioxidative effects of the Chinese prescription Kangen-karyu and its crude drug component Carthami Flos. The administration of Kangen-karyu extract at 100 mg/kg body weight per day for 10 weeks inhibited generation of nitric oxide, superoxide and the hydroxyl radical (*OH), while Carthami Flos extract showed only *OH-scavenging activity. Diet supplemented with Kangen-karyu and Carthami Flos extracts enhanced the activities of the antioxidative enzymes superoxide dismutase in hepatic tissue and glutathione peroxidase in renal tissue, and reduced the hepatic lipid peroxidation level which increased with aging, indicating the protective action against oxidative stress by enhancing the antioxidative status. Hepatic and renal dysfunction with aging was also ameliorated by the administration of Kangen-karyu and Carthami Flos supplements. Furthermore, the observed antioxidative properties of the Chinese prescription Kangen-karyu were more evident than those of Carthami Flos. These findings suggest that the protective activity of Kangen-karyu against the oxidative tissue damages during aging may be due partly to synergistic and/or additive effects of its crude preparation. The present study strongly indicates that Kangen-karyu counteract the oxidative stress and ameliorating tissue damage possibly associated with aging in SAM.