Determination of hydrogen peroxide scavenging activity of cinnamic and benzoic acids employing a highly sensitive peroxyoxalate chemiluminescence-based assay: structure-activity relationships

A series of cinnamic acids along with their corresponding benzoate analogues were tested for their ability to scavenge hydrogen peroxide (H(2)O(2)), by using a highly sensitive, peroxyoxalate chemiluminescence assay. Among benzoic acid derivatives, vanillic acid (3-hydroxy-4-methoxybenzoic acid) was found to be the most efficient H(2)O(2) scavenger with its hydrogen peroxide scavenging activity (SA(HP)) being 170.20 microM(-1), whereas protocatechuic acid (3,4-dihydroxybenzoic acid) exhibited the weakest activity (5.90 microM(-1)). Caffeic acid (3,4-dihydroxycinnamic acid) was the strongest antioxidant amongst cinnamate derivatives with a SA(HP) = 8.2 microM(-1), as opposed to m-coumaric acid (2-hydroxycinnamic acid), which was found to be a poor hydrogen peroxide scavenger (SA(HP) = 0.18 microM(-1)). Comparison between the two groups revealed that benzoate derivatives are much stronger hydrogen peroxide quenchers in relation with their cinnamate analogues, and this finding was discussed on a basis of structure-activity relationships and comparative assessment of other antioxidant characteristics.     

咖啡酸对UVB损伤人角质形成细胞的保护作用机制研究

目的 分析咖啡酸对UVB损伤人角质形成细胞HaCaT的保护作用机制。方法 采用UVB灯照射建立HaCaT细胞UVB损伤模型。HaCaT细胞中分别加入浓度梯度为0（对照组）、5、10、20、40、80、160μmol/L的咖啡酸,计算细胞存活率,选择最佳浓度进行后续试验。将处于对数生长期HaCaT分为对照组、模型组、咖啡酸（10μmol/L）组,进行苏木精–伊红（HE）染色,显微镜观察细胞形态。按照试剂盒说明书操作,采用样本蛋白浓度计算方法,测定细胞中过氧化氢酶（CAT）、超氧化物歧化酶（SOD）的含量。蛋白免疫印记检测促分裂原活化蛋白激酶（MAPK）通路相关蛋白的表达。结果咖啡酸10μmol/L组能够减轻UVB对HaCaT细胞造成的损伤,修复HaCaT的细胞形态,增加抗凋亡蛋白Bcl-2在HaCaT细胞中的表达,减少细胞的凋亡。咖啡酸10μmol/L组能够显著升高HaCaT中SOD、CAT含量,增强细胞中的抗氧化能力,降低MAPK亚族p38信号通路p-p38和p53蛋白在细胞中的表达。结论 咖啡酸可以抑制UVB对细胞的损伤,修复细胞的形态结构,减少细胞的凋亡和增强细胞的抗氧化性,且可能...     

Characterization of the radical scavenging and antioxidant activities of danshensu and salvianolic acid B.

Danshensu (3-(3,4-dihydroxyphenyl) lactic acid) and salvianolic acid B, two natural phenolic acids of caffeic acid derivatives isolated from Salvia miltiorrhiza root of the most widely used traditional Chinese medicine for the treatment of various cardiovascular diseases, have been reported to have potential protective effects from oxidative injury. To better understand their biological functions, the in vitro radical scavenging and antioxidant activities of danshensu and salvianolic acid B were evaluated along with vitamin C. Both danshensu and salvianolic acid B exhibited higher scavenging activities against free hydroxyl radicals (HO()), superoxide anion radicals (O(2)(-)), 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals and 2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals than vitamin C. In contrary, danshensu and salvianolic acid B showed weaker iron chelating and hydrogen peroxide (H(2)O(2)) scavenging activities than vitamin C. As expressed as vitamin C equivalent capacity (VCEAC), the relative VCEAC values (mg/100ml) were in the order of salvianolic acid B (18.59) > danshensu (12.89) > vitamin C (10.00) by ABTS radical assay. The protective efficiencies against hydrogen peroxide induced human vein vascular endothelial cell damage were correlated with their antioxidant activities. Analysis of structure-activity relationship of these two compounds showed that the condensation and conjugation of danshensu and caffeic acid appears important for antioxidant activity. These results indicated that danshensu and salvianolic acid B are efficient radical scavengers and antioxidants, and salvianolic acid B is superior to danshensu. Their radical scavenging and antioxidant properties might have potential applications in food and healthcare industry.     

Chroman/catechol hybrids: synthesis and evaluation of their activity against oxidative stress induced cellular damage

Three series of chromans substituted at positions 2 or 5 by catechol derivatives were synthesized, and their activity against oxidative stress induced cellular damage was studied. Specifically, the ability of the new molecules to protect cultured cells from H(2)O(2)-induced DNA damage was evaluated using single cell gel electrophoresis (comet assay), while the neuroprotective activity of the new compounds against oxidative stress induced programmed cell death was studied using glutamate-challanged hippocampal HT22 cells. The majority of the new compounds are stronger neuroprotectants than quercetin. 5-Substituted chroman analogues such as the caffeic acid amides 12 and 16 and the dihydrostilbene analogue 24 were the most potent against both H(2)O(2)- and glutamate-induced damage in Jurkat T cells and HT22 cells, respectively.     

Neuropharmacological analysis of caffeic acid in rats

The aim of the present study was to investigate the effect of intraperitoneal administration of caffeic acid (0.5, 1, 2, 4 or 8 mg/kg) on elevated plus-maze and open field tasks in rats and its possible neuroprotection/neurotoxicity using the comet assay. Caffeic acid at 1 mg/kg increased the number of entries and the time spent in the open arms on plus-maze, suggesting an anxiolytic-like effect when used in lower doses without affecting locomotion and exploration on the open field. Furthermore, a protective effect against hydrogen peroxide-induced oxidative damage on brain tissue was observed through the treatment with caffeic acid at 1 and 8 mg/kg. However, in the highest dose, caffeic acid induced DNA damage in brain tissue.     

Anti-oxidative and anti-aging activities of 2-O-α-glucopyranosyl-L-ascorbic acid on human dermal fibroblasts

A stable ascorbic acid derivative, 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G), was evaluated and compared with ascorbic acid for its protective effect against cellular damage and senescence induced by hydrogen peroxide (H(2)O(2)). Pretreatment with AA-2G for 72 h promoted the proliferation of normal human dermal fibroblasts (NHDF) and protected against cell damage induced by H(2)O(2). In contrast, ascorbic acid increased the proliferation and protected against cell damage, only when culture medium containing ascorbic acid was replaced every 24 h during the pretreatment period. These results suggest that the effect of AA-2G is longer-lasting compared to that of ascorbic acid. Senescence associated-β-galactosidase (SA-β-gal) activity, a classical biomarker of cellular senescence, was increased in H(2)O(2)-exposed NHDF cells, but pretreatment or posttreatment with ascorbic acid or AA-2G significantly inhibited the increase in SA-β-gal levels. AA-2G was more potent than ascorbic acid in down-regulating SA-β-gal activity. Expression of SIRT1, which has attracted attention as an anti-aging factor in recent years, was significantly decreased in H(2)O(2)-exposed NHDF cells compared to untreated cells. However, pretreatment NHDF cells with AA-2G before H(2)O(2) exposure significantly inhibited this decrease in SIRT1 expression, whereas ascorbic acid had no effect. After H(2)O(2) exposure, the expression levels of p53 and p21 were increased in NHDF cells and pretreatment with AA-2G inhibited this increase. Together, these results suggest that AA-2G protects dermal fibroblasts from oxidative stress and cellular senescence. These characteristics indicate that AA-2G could become a promising material for its anti-aging properties.     

Differential activity of lipoic acid enantiomers in cell culture

It is unclear whether the two enantiomeric forms (R & S) of lipoic acid (LA) share similar pharmacological activity and the exact cellular targets of LA are not well identified. We oxidatively stressed 3 cell culture systems representing different cell types. Mitochondrial metabolism was the primary endpoint. When C6 glioma was damaged by hydrogen peroxide (H2O2), all forms of LA protected. Racemic and S-LA were less effective than the R-isomer that was also protective in tertiary butyl hydroperoxide (TBHP)-damaged C6 glioma. In PC12 cells, little damage was produced by TBHP; R-LA increased mitochondrial metabolism above the level of non-damaged control. In H2O2 damaged PC12 cells, R-LA and racemic LA (but not S-LA) not only protected against damage, but increased mitochondrial metabolism above the non-damaged control level. When BAE cells were damaged with H2O2, R- and racemic LA protected while S-LA was ineffective.     

Preventive and therapeutic effects of caffeic acid against inflammatory injury in striatum of MPTP-treated mice

Preventive or therapeutic effects of caffeic acid in brain of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mice against inflammatory injury were examined. Caffeic acid at 0.5, 1 or 2% was supplied either pre-intake or post-intake for 4 weeks. Brain caffeic acid content was increased by caffeic acid pre-intake at 1 and 2%, and post-intake at 2% (P < 0.05). MPTP treatment enhanced the release of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, IL-4 and IL-10 (P < 0.05). Pre-intake of caffeic acid decreased the production of test cytokines (P < 0.05); however, post-intake only at 2% reduced the level of IL-1beta, IL-6 and TNF-alpha (P < 0.05). MPTP treatment up-regulated mRNA expression of inducible nitric oxide synthase (iNOS), neuronal NOS, cyclooxygenase (COX)-2, glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1, and increased production of nitric oxide (NO) and prostaglandin E? (PGE?) (P < 0.05). Caffeic acid pre-intake at test doses and post-intake at 2% declined the expression of iNOS, COX-2 and GFAP; and lowered the production of NO and PGE? (P < 0.05). MPTP treatment suppressed mRNA expression of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and tyrosine hydroxylase (TH), and lowered dopamine level (P < 0.05). Caffeic acid pre-intake retained the expression of these factors, maintained TH activity and protein production, and dopamine synthesis (P < 0.05). These results suggest that caffeic acid is a potent neuroprotective agent against the development of Parkinson's disease.     

Cytoprotective effect of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, on oxidative stress induced by hydrogen peroxide

In the present study, the antioxidative properties of tectorigenin, a metabolite formed by transformation of tectoridin by intestinal microflora, were investigated. Tectorigenin was found to scavenge intracellular reactive oxygen species, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and thus prevented lipid peroxidation. The radical scavenging activity of tectorigenin protected the viability of Chinese hamster lung fibroblast (V79-4) cells exposed to hydrogen peroxide (H2O2) via activation of extracellular signal regulated kinase (ERK) pathway. Furthermore, tectorigenin reduced the apoptotic cells formation and cell cycle arrest at G2/M phase induced by H2O2. Tectorigenin increased the activities of cellular antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase, and also increased their protein level. Taken together, these findings suggest that tectorigenin protected V79-4 cells against H2O2 damage, by enhancing the antioxidative activity and by activating ERK pathway.     

丹酚酸A对过氧化氢诱导的人脐静脉内皮细胞损伤的保护作用

目的研究丹酚酸A对人脐静脉内皮细胞(HUVECs)氧化损伤的保护作用。方法采用过氧化氢(H2O2)诱导的HUVECs氧化损伤模型,观察丹酚酸A(10,20和40μg·mL-1)对血管内皮细胞损伤的保护作用,MTT法检测细胞存活率,试剂盒检测细胞上清液中的超氧化物歧化酶(SOD)、丙二醛(MDA)、乳酸脱氢酶(LDH)、一氧化氮(NO),比色法测定Caspase-3的活性。结果丹酚酸A可抑制H2O2诱导的血管内皮细胞的凋亡,同时增加SOD的活性,降低MDA的水平,减少LDH的漏出量,增加NO的释放,降低Caspase-3的活性。且丹酚酸A的上述作用均呈质量浓度依赖性。结论丹酚酸A可以有效保护内皮细胞免受氧化损伤。     

Protective effect of protocatechuic acid from Alpinia oxyphylla on hydrogen peroxide-induced oxidative PC12 cell death

The neuroprotective effects of protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, on hydrogen peroxide (H(2)O(2))-induced apoptosis and oxidative stress in cultured PC12 cells were investigated. Exposure of PC12 cells to 0.4 mM H(2)O(2) induced a leakage of lactate dehydrogenase (LDH) and decreased cell viability denoted by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PCA increased PC12 cellular viability and markedly attenuated H(2)O(2)-induced apoptotic cell death in a dose-dependent manner. By flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against H(2)O(2)-induced apoptosis. In these cells, the levels of glutathione (GSH) and activity of catalase were augmented, while glutathione peroxidase activity remained unchanged. In addition, PCA also protected against cell damage induced by H(2)O(2) and Fe(2+), which generated hydroxyl radicals (OH) by the Fenton reaction. These results suggest that PCA may be a candidate chemical for the treatment of oxidative stress-induced neurodegenerative disease.     

Catalase activity in coronary artery endothelium protects smooth muscle against peroxide damage

Cyclopiazonic acid contracts pig coronary artery de-endothelialized rings, and pretreating the rings with hydrogen peroxide (H(2)O(2)) inhibits this contraction (IC(50)=0.097+/-0.013 mM). We used the cyclopiazonic acid contraction to test the novel hypothesis that endothelium can protect underlying smooth muscle against luminal H(2)O(2). We perfused the arteries with Krebs' solution containing 0. 3 or 1 mM H(2)O(2), removing endothelium from the arteries either before or after the perfusion. We then cut rings from them to monitor their contraction to 10 microM cyclopiazonic acid in a H(2)O(2)-free solution. The inhibition of the cyclopiazonic acid contraction by perfusion with H(2)O(2) was significantly less when endothelium was removed after the perfusion than when it was removed before it. The specific activity of catalase in post-nuclear supernatants from freshly isolated endothelium (14.1+/-2.7 micromol/min/mg protein) was 17+/-3-fold greater than in those from smooth muscle (0.83+/-0.22 micromol/min/mg protein). Thus endothelium contained high catalase activity and protected the underlying smooth muscle against luminal peroxide.     

Ascorbic acid induces redifferentiation and growth inhibition in human hepatoma cells by increasing endogenous hydrogen peroxide

The mechanisms of redifferentiation and growth inhibition induced in human hepatoma cells by ascorbic acid (AA) were studied. After treatment with AA, the content of hydrogen peroxide (H2O2) and the activity of superoxide dismutase (SOD) increased in a concentration- and time-dependent manner, while the activity of catalase (CAT) decreased in a concentration- and time-dependent manner. Using 6 mM AA as a positive control, after treatment by 50 microM hydrogen peroxide, the malignant characteristics of human hepatoma cells were alleviated; for example as cell surface charge markedly decreased, the electrophoresis rate dropped from 1.68 microns.s-1.V-1.cm-1 to 0.97, the average of alpha-fetoprotein content decreased from 327 micrograms.g-1 protein to 193, and gamma-glutamyl-transpeptidase activity fell from 0.84 U.g-1 protein to 0.30. The indexes related to cell differentiation were promoted, such as tyrosine-alpha-ketoglutarate transaminase activity increased from 17.1 mumol.g-1 protein to 33.1, and the colonogenic potential decreased by 79.3%. SOD and 3-amino-1,2,4-triazole (AT) exhibited some effects, but there were statistically significant differences between the SOD, AT and H2O2 or AA groups. AA induced growth inhibition and redifferentiation of human hepatoma cells through the production of hydrogen peroxide, since addition of SOD (200 units/ml), an enzyme that dismutates superoxide and generates hydrogen peroxide, and AT (1.5 mM), a CAT inhibitor that inhibits the activity of CAT and leads to an increase in H2O2 content, showed some inducing changes emphasizing the involvement of reactive oxygen species (ROS) in redifferentiation of hepatoma cells. AA can cause the content of H2O2 to increase, and the factor H2O2 showed a similar effect to AA on growth and redifferentiation suggests that H2O2 is involved in hepatoma cell redifferentiation. In conclusion, these results suggest that AA inhibits tumor growth and induces tumor redifferentiation by virtue of producing H2O2.     

Myricetin suppresses oxidative stress-induced cell damage via both direct and indirect antioxidant action

We evaluated the cytoprotective effect of myricetin on oxidative stress damaged cells by assessment of the scavenging effect of reactive oxygen species (ROS) and the activities of antioxidant enzymes. Myricetin showed the scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals on intracellular ROS. In addition, myricetin restored the activity and protein expression of cellular antioxidant defense enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) reduced by hydrogen peroxide (H(2)O(2)) treatment. H(2)O(2)-induced cellular DNA and lipid damages, and myricetin was found to prevent the DNA damage shown by inhibition of DNA tail and it decreased nuclear phospho-histone H2A.X expression, which are both markers for DNA strand breakage. Membrane lipid peroxidation was also attenuated as shown by inhibition of TBARS formation and of fluorescence intensity of diphenyl-1-pyrenylphosphine (DPPP). These results suggest that myricetin protects cells against H(2)O(2)-induced cell damage via inhibition of ROS generation and activation of antioxidant enzymes.     

Protective effect of erdosteine metabolite I against hydrogen peroxide-induced oxidative DNA-damage in lung epithelial cells

It has been shown that the mucolytic agent erdosteine (N-carboxymethylthio-acetyl-homocysteine thiolactone, CAS 84611-23-4) has anti-inflammatory and anti-oxidant properties, and an active metabolite I (MET I) containing pharmacologically active sulphydryl group has been found to have a free radical scavenging activity. The aim of this study was to assess the ability of erdosteine metabolite I to protect A549 human lung adenocarcinoma cell against hydrogen peroxide (H2O2)-mediated oxidative stress and oxidative DNA damage. When A549 cells were pre-treated with the active metabolite I (2.5-5-10 microg/ml) for 10-30 min and then exposed to H2O2 (1-4 mM) for two additional hours at 37 degrees C, 5% at CO2, the intracellular peroxide production, reflected by dichlorofluorescein (DCF) fluorescence, decreased in a concentration-dependent manner. Furthermore, using a comet assay as an indicator for oxidative DNA damage, it was found that the metabolite I prevented damage to cells exposed to shortterm H2O2 treatment. The data suggest that this compound is effective in preventing H2O2-induced oxidative stress and DNA damage in A549 cells. The underlying mechanisms involve the scavenging of intracellular reactive oxygen species (ROS).     

Protective effect of the methanolic extract from Duchesnea indica against oxidative stress in vitro and in vivo

Duchesnea indica (Rosaceae family) is herb used extensively in traditional Chinese medicine. In this study we investigated its protective activity against hydrogen peroxide (H(2)O(2))-induced cytotoxicity in human skin fibroblast (CCD-986Sk) cells and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced H(2)O(2) in the skin of hairless mice. Pretreatment of CCD-986Sk cells with methanolic extract of D. indica (DIM) improved the cell viability, enhanced activity of catalase, and decreased the leakage of lactate dehydrogenase (LDH) and the levels of malondialdehyde (MDA) and intracellular reactive oxygen species (ROS) in H(2)O(2) injured cells. Furthermore, DIM inhibited cell apoptosis and Bax expression induced by H(2)O(2). In addition, the level of H(2)O(2) stimulated by TPA was decreased by DIM in the skin of hairless mice. These results suggest that DIM offers protection against oxidative stress in vitro and in vivo, and this ability suggests potential use for protection against oxidation-induced skin damage.     

Antioxidant phenolic profile from ethyl acetate fraction of Fructus Ligustri Lucidi with protection against hydrogen peroxide-induced oxidative damage in SH-SY5Y cells

In this study, we demonstrated the antioxidant and protective properties of crude extract and fractions from Fructus Ligustri Lucidi (FLL) against hydrogen peroxide (H2O2)-induced oxidative damage in SH-SY5Y cells. The contents of their phytochemical profiles were determined by spectrophotometric methods and high performance liquid chromatography using a photodiode array detector. FLL crude extract possessed appreciable scavenging capacity against 1,1-diphenyl-2-picrylhydrazyl and H2O2. The ethyl acetate (EtOAc) fraction was the most active fraction in scavenging free radicals and H2O2. Following exposure of cells to H2O2, there was a marked decrease in cell survival and intracellular antioxidant enzymes, and then intracellular oxidative stress, the level of lipid peroxidation, and caspase-3 activity were increased. Simultaneous treatment with the EtOAc fraction blocked these H2O2-induced cellular events. Hydroxytyrosol and salidroside are major components of the EtOAc fraction. These results show that the phenolic-enriched EtOAc fraction of FLL contains tyrosol-related derivatives and exerts the protective effects against H2O2 toxicity via its free radical scavenging activity and ability to elevate the levels of antioxidant enzymes.