Zhonghong Gao

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten micromol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe(2+)-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 micromol/l of baicalein and baicalin significantly protected cells against H(2)O(2)-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.

Hypoglycaemic effect of a novel INSULIN BUCCAL formulation on rabbits

Transmucosal delivery is a suitable route for insulin non-injection administration. In this study, the hypoglycaemic effect of INSULIN BUCCAL SPRAY (IBS), a formulation with soybean lecithin and propanediol combined as absorption enhancer for insulin on diabetic rabbits and rats, were investigated. The hypoglycaemic rate was calculated and the pharmacodynamics and pharmacokinetics of the formulation in rabbits were studied. The results show that when the diabetic rabbits were administrated with IBS in dosages of 0.5, 1.5 and 4.5Ukg(-1), the blood glucose level decreased significantly compared with that of the control group and the hypoglycaemic effect lasted over 5h. The blood glucose decreasing rates are 22.4, 48.1 and 53.5%, respectively. The average bioavailability of IBS by buccal delivery versus subcutaneous injection is 29.2%. Meanwhile, the diabetic rats were administrated with IBS in dosages of 1.0, 3.0 and 9.0Ukg(-1), the blood glucose level decreased significantly compared with that of the control group and the hypoglycaemic effect lasted over 4h. The blood glucose decreasing rates are 24.6, 47.5 and 59.6%, respectively. Furthermore, the penetration of fluorescein isothiocyanate (FITC)-labelled insulin through rabbit buccal mucosa was investigated by scanning the distribution of the fluorescent probe in the epithelium using confocal laser scanning microscopy. The results revealed that FITC-insulin can pass through the buccal mucosa promoted by the enhancer and the passage of insulin across the epithelium includes both intracellular and paracellular routes. From the rabbit and rat experimental results showed that IBS is an effective buccal delivery system, which is promising for clinical trial and the future clinical application.

Effects of rutin supplementation on antioxidant status and iron, copper, and zinc contents in mouse liver and brain.

The effect of rutin on total antioxidant status as well as on trace elements such as iron, copper, and zinc in mouse liver and brain were studied. Mice were administrated with 0.75 g/kg or 2.25 g/kg P. O. of rutin for 30 d consecutively. Following the treatment, the activity of total antioxidant status, catalase, Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, zinc, copper, and iron were measured in mouse liver and brain. The results showed that rutin significantly increased the antioxidant status and Mn-superoxide dismutase activities in mouse liver, but it had no effect on these variables in the brain. Treatment with a higher concentration of rutin significantly decreased catalase activity and iron, zinc, and copper contents in mouse liver; it also resulted in a slower weight gain for the first 20 d. These results indicate that rutin taken in proper amount can effectively improve antioxidant status, whereas at an increased dosage, it may cause trace element (such as iron, zinc, and copper) deficiencies and a decrease in the activities of related metal-containing enzymes.

Iron overload-induced rat liver injury: Involvement of protein tyrosine nitration and the effect of baicalin.

Baicalin has been reported to protect against liver injury in iron-overload mice, however, the mechanisms underlying the hepatoprotective properties of baicalin are poorly understood. In this study, we systematically studied the protective effect of baicalin on iron overload induced liver injury, as well as the underlying mechanism based on nitrative stress in rat model. We found that when iron overload rats (500mgiron/kg) were fed baicalin-containing diet (0.3% and 1% w/w) for 45days, baicalin dose dependently protected against iron overload induced liver injury, including alleviation of hepatic pathological damage, decrease of SOD activity, iron content, carbonyl content, and the thiobarbituric acid-reactive substances level in hepatic tissues. It also increased serum iron content, SH content and GPx activity, decreased serum ALT and AST activities. Immunohistochemistry and immunoprecipitation analysis revealed that baicalin could also inhibit iron overload induced protein tyrosine nitration in liver. Moreover, in iron overload rat liver, we found that baicalin decreased the iron overload increased level of glutathione-S-transferases (GSTs) expression, oxidation and nitration. These results suggest that not only oxidative stress, but also nitrative stress, is involved in iron overload induced liver injury, and the underlying mechanism might partially relate to the involvement of GSTs expression and post-translational modification. Baicalin can effectively prevent iron overload caused abnormality and can be a candidate medicine for iron overload diseases.

Iron increases liver injury through oxidative/nitrative stress in diabetic rats: Involvement of nitrotyrosination of glucokinase.

Excessive tissue iron levels are associated with the increase of oxidative/nitrative stress which contributes to tissue damage that may elevate the risk of diabetes. Therefore, we investigated the effects of iron on diabetes-associated liver injury and whether iron-related tyrosine nitration participated in this process. Rats were randomly divided into four groups: control, iron overload (300 mg/kg iron dextran, i.p.), diabetic (35 mg/kg of streptozotocin i.p. after administration of a high-fat diet) and diabetic simultaneously treated with iron. Iron supplement markedly increased diabetes-mediated liver damage and hepatic dysfunction by increasing liver/body weight ratio, serum levels of aspartate and alanine aminotransferase, and histological examination, which were correlated with elevated levels of lipid peroxidation, protein carbonyls and tyrosine nitration, oxidative metabolism of nitric oxide, and reduced antioxidant capacity. Consequently, the extent of oxidized/nitrated glucokinase was markedly increased in the iron-treated diabetic rats that contribute to a decrease in its expression and activity. Further studies revealed a significant contribution of iron-induced specific glucokinase nitration sites to its inactivation. In conclusion, iron facilitates diabetes-mediated elevation of oxidative/nitrative stress, simultaneously impairs liver GK, and can be a link between enzymatic changes and hepatic dysfunction. These findings may provide new insight on the role of iron in the pathogenesis of diabetes mellitus.

Amyloid beta–heme peroxidase promoted protein nitrotyrosination: relevance to widespread protein nitration in Alzheimer’s disease

Amyloid beta (Aβ) peptide accumulation has been demonstrated to play a central role in Alzheimer’s disease (AD). Substantial evidence indicates that protein nitrotyrosination contributes to Aβ-dependent neurotoxicity; however, the molecular mechanism is unknown. Recent research has shown that Aβ complexes with heme to form Aβ–heme, and increases the pseudo-peroxidase activity of heme. We found that Aβ–heme uses H2O2 and NO2− to cause nitration of enolase and synaptic proteins more effectively than heme. Thus, the increased peroxidase activity of Aβ–heme may be the molecular link between excess Aβ and the widespread protein nitration in AD. Interestingly, the site of enolase nitration that was catalyzed by Aβ–heme is different from that induced by heme. Moreover, the secondary structural perturbations of Aβ–heme-treated and heme-treated enolase are also different. These observations suggest that Aβ–heme targets specific amino acid sequences in enolase. Furthermore, our data show that Aβ–heme peroxidase activity is independent of the aggregation state of Aβ, suggesting an important role of soluble Aβ in addition to Aβ aggregates and oligomers in AD pathogenesis.

Free-radical scavenging and mechanism study of flavonoids extracted from the radix ofScutellaria baicalensis Georgi

Free-radical scavenging activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in a traditional Chinese herb medicine, the radix ofScutellaria baicalensis Georgi, were examined by electron paramagnetic resonance (EPR). The results showed that baicalein and baicalin scavenged hydroxyl radical, superoxide anion, 2,2-diphenyl-1-picrylhydrazyl radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Baicalein was the most effective free-radical scavenger among the four tested compounds. When 10 mmol/l tested flavonoids dissolved in alkaline solution, only baicalein and baicalin form stable free radicals which can be detected by EPR technique, it was found that the signal came from theiro-di-hydroxyl structure in ring A. To our knowledge, it was the first time demonstrated that flavonoids witho-di-hydroxyl structure in ring A could also form stable semiquinone free radicals. These results demonstrated that flavonoids in radix ofScutellaria baicalensis witho-di-hydroxyl group in ring A such as baicalein and baicalin are good free-radical scavengers and might contribute to some of their pharmaceutical effects.

Aqueous Two-Phase System as an Effective Tool for Purification of Phenolic Compounds from Fig Fruits (Ficus carica L.)

Fig fruits (Ficus carica L.) are an excellent source of phenolic compounds. However, the presence of free sugars affects the quality of phenolic compounds. In this study, aqueous two-phase system was selected for purification of the phenolic compounds. The optimization of system parameters rendered conditions (ethanol 18% (w/w), K2HPO4 25% (w/w), temperature 10-30ºC, and sample loading 3% (w/w)), under which more than 75% of phenolic compounds and 95% sugars were respectively recovered in the top and bottom phase. Moreover, some specific phenolic compounds content was enhanced and relatively high antioxidant activity was found after the purification process.

The stress caused by nitrite with titanium dioxide nanoparticles under UVA irradiation in human keratinocyte cell

Our previous work found that in the presence of nitrite, titanium dioxide nanoparticles can cause protein tyrosine nitration under UVA irradiation in vivo. In this paper, the human keratinocyte cells was used as a skin cell model to further study the photo-toxicity of titanium dioxide nanoparticles when nitrite was present. The results showed that nitrite increased the photo-toxicity of titanium dioxide in a dose-dependant manner, and generated protein tyrosine nitration in keratinocyte cells. Morphological study of keratinocyte cells suggested a specific apoptosis mediated by apoptosis inducing factor. It was also found the main target nitrated in cells was cystatin-A, which expressed abundantly in cytoplasm and functioned as a cysteine protease inhibitor. The stress induced by titanium dioxide with nitrite under UVA irradiation in human keratinocyte cells appeared to trigger the apoptosis inducing factor mediated cell death and lose the inhibition of active caspase by cystatin-A. We conclude that nitrite can bring new damage and stress to human keratinocyte cells with titanium dioxide nanoparticles under UVA irradiation.

Iron increases diabetes-induced kidney injury and oxidative stress in rats.

Diabetic nephropathy is both a common and a severe complication of diabetes mellitus. Iron is an essential trace element. However, excess iron is toxic, playing a role in the pathogenesis of diabetic nephropathy. The present study aimed to determine the extent of the interaction between iron and type 2 diabetes in the kidney. Male rats were randomly assigned into four groups: control, iron (300-mg/kg iron dextran), diabetes (a single dose of intraperitoneal streptozotocin), and iron + diabetes group. Iron supplementation resulted in a higher liver iron content, and diabetic rats showed higher serum glucose compared with control rats, which confirmed the model as iron overload and diabetic. It was found that iron + diabetes group showed a greater degree of kidney pathological changes, a remarkable reduction in body weight, and a significant increase in relative kidney weight and iron accumulation in rat kidneys compared with iron or diabetes group. Moreover, malondialdehyde values in the kidney were higher in iron + diabetes group than in iron or diabetes group, sulfhydryl concentration and glutathione peroxidase activity were decreased by the diabetes and iron + diabetes groups, and protein oxidation and nitration levels were higher in the kidney of iron + diabetes group as compared to iron or diabetes group. However, iron supplementation did not elevate the glucose level of a diabetic further. These results suggested that iron increased the diabetic renal injury probably through increased oxidative/nitrative stress and reduced antioxidant capacity instead of promoting a rise in blood sugar levels; iron might be a potential cofactor of diabetic nephropathy, and strict control of iron would be important under diabetic state.