Rhodiola rosea as antioxidant in red blood cells: ultrastructural and hemolytic behaviour

Rhodiola rosea L. (Crassulaceae) is a plant that lives at high altitude in Europe and Asia, widely used for its high capacity to increase the organism resistance to different stress conditions. Although a few international literature supports these effects, today R. rosea has become a common component of many dietary supplements also in the Western world. The aim of the present study was to investigate the effect of the R. rosea roots aqueous extract on in vitro human erythrocytes exposed to hypochlorous acid (HOCl)-oxidative stress. Several damages occur in human erythrocytes exposed in vitro to HOCl, among these membrane protein and lipid modifications, shifting from the discocyte shape to the echinocyte one, and determining lysis ultimately. Therefore, in the present work, the evaluation of the antioxidant capacity of the Rhodiola extract has been carried out by means of scanning electron microscopy and of hemolytic behaviour on human erythrocytes exposed to HOCl in the presence of increasing doses of the aqueous extract in different experimental environments (co-incubation and subsequent incubations). The results obtained are consistent with a significant protection of the extract in presence of the oxidative agent, but a cautionary note emerges from the analysis of the data related to the cell exposition to the plant extract in the absence of any induced oxidative stress. In fact, the addition to erythrocyte of high doses of R. rosea extract always determines severe alterations of the cell shape.     

Serotonin involvement in Rhodiola rosea attenuation of nicotine withdrawal signs in rats

Rhodiola rosea has been used for centuries in the traditional medicine to stimulate nervous system, to enhance physical and mental performance and to treat fatigue. It is known that administration of Rhodiola rosea extract elicits antidepressant activity, but the mechanism of action still remains unclear. Evidence from animal models and human studies show that nicotine reduces symptoms of depression and that nicotine cessation induces depressive-like symptoms. We investigated the effects of Rhodiola rosea on nicotine withdrawal signs. Nicotine dependence was induced by subcutaneous nicotine injection (2mg/kg, four times daily) for 14 days. Another group of animals treated with nicotine (for 14 days) and successively with Rhodiola rosea extract was co-administered with selective 5-HT receptorial antagonist WAY 100635 (1mg/kg). After nicotine withdrawal animals were evaluated for behavioural parameters (locomotor activity, abstinence signs, marble burying test), diencephalic serotonin metabolism and serotonin receptor-1A expression. Results show a significant increase of 5-HT content in N treated with R. rosea, with a significant increase of serotonin receptor 1A, suggesting an involvement of serotonin in beneficial effects of R. rosea on suffering produced by nicotine withdrawal.     

红景天提取物提高高强度跑台运动小鼠的抗氧化能力

为了考察红景天提取物对高强度跑台运动小鼠的抗氧化能力的影响,本研究将30只昆明小鼠随机分成对照组、模型组和红景天提取物组,每组10只。红景天提取物组小鼠按照500 mg/kg bw的剂量灌胃红景天提取液(2 m L)。对照组和模型组小鼠灌胃等体积的蒸馏水,共灌胃4周。采用硫酸蒽酮比色法检测小鼠肝脏和肌肉组织糖原的含量;采用硫代巴比妥酸比色法检测小鼠骨骼肌组织中的丙二醛(MDA)水平;采用RT-PCR和Western blotting检测骨骼肌组织中的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)和过氧化氢酶(CAT)的表达水平;采用苏木精和伊红(HE)染色评价骨骼肌病理改变。研究显示,模型组小鼠的跑台运动时间显著低于红景天提取物组(61.32 min vs 83.22 min,p<0.05);与模型组相比,红景天提取物组小鼠骨骼肌的炎性细胞浸润明显减轻,肌纤维排列明显改善;红景天提取物组的肝糖原和肌糖原含量均显著高于模型组;红景天提取物组小鼠骨骼肌组织中的MDA水平显著低于模型组;红景天提取物组的SOD、GSH-Px和CAT m RNA和蛋白表达水平均显著高于模型组;红景天提取物可通过上调抗氧化酶表达来增加抗氧化能力,减弱骨骼肌损伤,并增加机体的抗疲劳能力。     

Protection of oxidative damage by aqueous extract from Antrodia camphorata mycelia in normal human erythrocytes

Antrodia camphorata (A. camphorata) is well known in Taiwan as a traditional Chinese medicine. The purpose of this study was to evaluate the ability of aqueous extract from A. camphorata mycelia to protect normal human erythrocytes against oxidative damage in vitro. Oxidative hemolysis and lipid/protein peroxidation of erythrocytes induced by the aqueous peroxyl radical [2,2'-Azobis(2-amidinopropane) dihydrochloride, AAPH] were suppressed by A. camphorata mycelia in a time-and concentration-dependent manner. A. camphorata mycelia also prevented the depletion of cytosolic antioxidant glutathione (GSH) and ATP in erythrocytes. Moreover, cultured human endothelial cell damage induced by AAPH was suppressed by A. camphorata mycelia. Interestingly, A. camphorata mycelia exhibited significant cytotoxicity against leukemia HL-60 cells but not against cultured human endothelial cells. These results imply that A. camphorata mycelia may have protective antioxidant and anticancer properties.     

The aqueous garlic, onion and leek extracts release nitric oxide from S-nitrosoglutathione and prolong relaxation of aortic rings

Garlic, onion and leek have beneficial effects in treatment of numerous health disorders. The aim of the present study was to investigate underlying molecular mechanisms. To test the potency of the aqueous garlic, onion and leek extracts to release NO from GSNO we have measured NO oxidation product, NO(2)-, by the Griess reagent method. Further, we studied the ability of garlic extract to relax noradrenaline-precontracted rat aortic rings in the presence of GSNO and effects of garlic extract on electrical properties of rat heart intracellular chloride channels. We have observed that: i) garlic, onion and leek extracts released NO from GSNO in the order: garlic > onion > leek; ii) the ability of garlic extract to release NO was pH-dependent (8.0 > 7.4 > 6.0) and potentiated by thiols (Cys > GSH = N-acetyl-cysteine > oxidized glutathione) at concentration 100 μmol/l; iii) the garlic extract (0.045 mg/ml) prolonged relaxation time of aortic rings induced by GSNO (50 nmol/l) and inhibited intracellular chloride channels. We suggest that NO-releasing properties of the garlic, onion and leek extracts and their interaction with Cys and GSH are involved in NO-signalling pathway which contributes to some of its numerous beneficial biological effects.     

Resveratrol affords protection against peroxynitrite-mediated endothelial cell death: A role for intracellular glutathione

Atherosclerosis, the main cause of cardiovascular disease (CD), is a chronic inflammatory condition associated with an overproduction of oxidant species, namely peroxynitrite, which is a powerful oxidant that reacts directly with all biomolecules. Glutathione is an efficient scavenger of peroxynitrite, so, modulation of glutathione synthesis may provide a strategy to selectively protect cells from this oxidant. Here, we investigated the ability of resveratrol, a component of red wine, to prevent peroxynitrite-mediated endothelial cells toxicity and the underlying mechanism. Bovine aortic endothelial cells (BAEC) in primary cultures were treated with authentic peroxynitrite and the cell viability and intracellular glutathione contents were assessed. Our results demonstrate that a long pre-incubation (14 h) of BAEC with resveratrol (1-50 microM) leads to the endothelial cells rescue from injury triggered by authentic peroxynitrite by a mechanism of up-regulation of the intracellular GSH content, for the highest resveratrol concentration tested. Considering the importance of GSH in regulation of cell life, this capacity of resveratrol provides a new mechanism for its cardioprotective effects and may contribute to the development of novel therapeutic strategies.     

Anti-inflammatory,antioxidant and antihepatotoxic effects of Spirulina platensis against d-galactosamine induced hepatotoxicity in rats

Spirulina platensis has been advocated as safe food for human use by several investigators. In this study its beneficial dietary effect against liver injuries caused by d-galactosamine (d-GalN) was studied ensuring safety to human health using animal model. Acute hepatotoxicity was induced in Wister rats with d-GalN followed by treatment with butylated hydroxytoluene (BHT) and with Spirulina aqueous extract at various concentrations. The effect of Spirulina at different concentrations were tried and compared with BHT treatment. The animals treated with d-GalN on subsequent treatment by supplementation with Spirulina (6, 9%) in the diets, led to significant reversal in the levels of the antioxidant enzymes through hepatocytes by suppression of negative effect. Spirulina aqueous extract at 9% resulted in a significant decrease in the levels of alkaline phosphatase and infalmatory markers TNFα, IL6 and IL1β and also decreased TBARS, while it showed an increase in oxidative stress marker such as GR, GSH, GST, SOD, GPX and CAT and total protein when compared to the levels recorded with that group treated with d-GalN. Results also indicated that Spirulina aqueous extract at 9% concentration was equally effective in protecting liver damage as it was observed with BHT. Histological studies on liver treated with d-GalN, BHT and Spirulina aqueous extract showed that S. platensis is effective as diet in providing beneficial protective effect. The results obtained in the present study very clearly indicated the positive beneficial protective effect of Spirulina, when used as diet, on the safety and protection of liver from injuries caused by toxicants.     

Effect of Aloe vera gel extract on antioxidant enzymes and azoxymethane-induced oxidative stress in rats

The present work was undertaken with a view to study the effect of oral feeding of 2% Aloe vera gel extract (AGE) for 30 days on azoxymethane (AOM)-induced oxidative stress in rats. It was observed that AOM administration resulted in a significant increase in malondialdehyde and conjugated dienes, with reduction in hepatic glutathione (GSH), vitamin A and uric acid contents. AOM-induced reduction in hepatic GSH and uric acid was brought back to normal by AGE. There was a significant raise in hepatic catalase, superoxide dismutase and glucose-6-phosphate dehydrogenase (G-6-PD) activities as a result of feeding of the extract. Ingestion of the extract effected reduction in AOM-induced colonic GSH-peroxidase, G-6-PD and glutathione S-transferase and femur bone marrow micronuclei formation. Hence, it is suggested that Aloe vera gel extract possess the ability to reduce AOM- induced oxidative stress and toxicity in liver.     

The potent pro‐oxidant activity of rhododendrol–eumelanin is enhanced by ultraviolet A radiation

RS‐4‐(4‐hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some consumers. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products and RD–eumelanin exerts a potent pro‐oxidant activity. Cellular antioxidants were oxidized by RD–eumelanin with a concomitant production of H₂O₂. In this study, we examined whether this pro‐oxidant activity of RD–eumelanin is enhanced by ultraviolet A (UVA) radiation because most RD–induced leukoderma lesions are found in sun‐exposed areas. Exposure to a physiological level of UVA (3.5 mW/cm²) induced a two to fourfold increase in the rates of oxidation of GSH, cysteine, ascorbic acid, and NADH. This oxidation was oxygen‐dependent and was accompanied by the production of H₂O₂. These results suggest that RD–eumelanin is cytotoxic to melanocytes through its potent pro‐oxidant activity that is enhanced by UVA radiation.     

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

Ascorbate uptake and antioxidant function in peritoneal macrophages

Since activated macrophages generate potentially deleterious reactive oxygen species, we studied whether ascorbic acid might function as an antioxidant in these cells. Thioglycollate-elicited murine peritoneal macrophages contained about 3 mM ascorbate that was halved by culture in ascorbate-free medium. However, the cells took up added ascorbate to concentrations of 6-8 mM by a high-affinity sodium-dependent transport mechanism. This likely reflected the activity of the SVCT2 ascorbate transporter, since its message and protein were present in the cells. Activation of the cells by phagocytosis of latex particles depleted intracellular ascorbate, although not below the basal levels present in the cells in culture. Glutathione (GSH) was unaffected by phagocytosis, suggesting that ascorbate was more sensitive to the oxidant stress of phagocytosis than GSH. Phagocytosis induced a modest increase in reactive oxygen species as well as a progressive loss of alpha-tocopherol, both of which were prevented in cells loaded with ascorbate. These results suggest that activated macrophages can use ascorbate to lessen self-generated oxidant stress and spare alpha-tocopherol, which may protect these long-lived cells from necrosis or apoptosis.     

Human erythrocyte recycling of ascorbic acid: relative contributions from the ascorbate free radical and dehydroascorbic acid

Recycling of ascorbic acid from its oxidized forms helps to maintain the vitamin in human erythrocytes. To determine the relative contributions of recycling from the ascorbate radical and dehydroascorbic acid, we studied erythrocytes exposed to a trans-membrane oxidant stress from ferricyanide. Ferricyanide was used both to induce oxidant stress across the cell membrane and to quantify ascorbate recycling. Erythrocytes reduced ferricyanide with generation of intracellular ascorbate radical, the concentrations of which saturated with increasing intracellular ascorbate and which were sustained over time in cells incubated with glucose. Ferricyanide also generated dehydroascorbic acid that accumulated in the cells and incubation medium to concentrations much higher than those of the radical, especially in the absence of glucose. Ferricyanide-stimulated ascorbate recycling from dehydroascorbic acid depended on intracellular GSH but was well maintained at the expense of intracellular ascorbate when GSH was severely depleted by diethylmaleate. This likely reflects continued radical reduction, which is not dependent on GSH. Erythrocyte hemolysates showed both NAD- and NADPH-dependent ascorbate radical reduction. The latter was partially due to thioredoxin reductase. GSH-dependent dehydroascorbate reduction in hemolysates, which was both direct and enzyme-dependent, was greater than that of the radical reductase activity but of lower apparent affinity. Together, these results suggest an efficient two-tiered system in which high affinity reduction of the ascorbate radical is sufficient to remove low concentrations of the radical that might be encountered by cells not under oxidant stress, with back-up by a high capacity system for reducing dehydroascorbate under conditions of more severe oxidant stress.     

Protective effect of aqueous garlic extract against oxidative organ damage in a rat model of thermal injury

Oxygen free radicals have been implicated in mediating various pathological processes including burn-induced organ damage. This study was designed to determine the possible protective effect of aqueous garlic extract against oxidative organ damage distant from the original burn wound. Under ether anaesthesia, rats were subjected to severe skin scald injury covering 30% of total body surface area. Rats were decapitated either 2 h or 24 h after burn injury. Aqueous garlic extract (1 ml/kg) was administered i.p. immediately after burn injury. In the 24-h burn group injection was repeated once more (at 12 hour) following the burn injury. Liver, intestine and lung tissues were taken for the determination of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity and protein oxidation (PO). Burn injury caused a significant decrease in GSH level, and significant increases in MDA and PO levels, and MPO activity at post-burn 2 and 24 hours. Since garlic extract reversed these oxidant responses it seems likely that garlic extract protects tissues against oxidative damage.     

Analgesic and anti-inflammatory activity of the aqueous extract of Rheedia longifolia Planch & Triana

Rheedia longifolia Planch et Triana belongs to the Clusiaceae family. This plant is widely distributed in Brazil, but its chemical and pharmacological properties have not yet been studied. We report here that leaves aqueous extract of R. longifolia (LAE) shows analgesic and anti-inflammatory effects. Oral or intraperitoneal administration of this extract dose-dependently inhibited the abdominal constrictions induced by acetic acid in mice. The analgesic effect and the duration of action were similar to those observed with sodium diclofenac, a classical non-steroidal analgesic. In addition to the effect seen in the abdominal constriction model, LAE was also able to inhibit the hyperalgesia induced by lipopolysaccharide from gram-negative bacteria (LPS) in rats. We also found that R. longifolia LAE inhibited an inflammatory reaction induced by LPS in the pleural cavity of mice. Acute toxicity was evaluated in mice treated with the extract for seven days with 50 mg/kg/day. Neither death, nor alterations in weight, blood leukocyte counts or hematocrit were noted. Our results suggest that aqueous extract from R. longifolia leaves has analgesic and anti-inflammatory activity with minimal toxicity and are therefore endowed with a potential for pharmacological control of pain and inflammation.     

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.     

Effect of Salvia miltiorrhiza on aflatoxin B1-induced oxidative stress in cultured rat hepatocytes

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B₁ (AFB₁). Salvia miltiorrhiza (Sm), a herbal plant that has been used extensively in traditional Chinese medicine for treating cardiovascular and liver diseases, is believed to have some antioxidative capabilities. In this study, the protective effect of Sm against AFB₁-induced cytotoxicity was investigated in cultured primary rat hepatocytes. AFB₁-induced cytotoxicity and lipid peroxidation (LPO) were estimated by determination of lactate dehydrogenase (LDH) leakage and thiobarbituric acid reactive substances (TBARS) formation, respectively. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). In addition, changes of intracellular glutathione (GSH) content were also studied. Results showed that Sm was able to suppress the LDH leakage induced by AFB₁ in a dose-dependent manner. A dose-dependent inhibitory effect of Sm on AFB₁-induced LPO was also found in hepatocytes treated with Sm. It was further observed that Sm produced an inhibitory effect on ROS formation caused by AFB₁. Concomitantly, the GSH content in Sm-treated groups increased substantially compared to those without Sm treatment. These findings suggest that Sm can inhibit the cytotoxicity of AFB₁ through decreasing ROS formation, inhibiting LPO and preventing GSH depletion. The major component of the aqueous extract of Sm was identified by using high performance liquid chromatography (HPLC), proton magnetic resonance (¹H-NMR) and mass spectrum (MS). Analytical results suggested that D(+)β3,4-dihydroxyphenol lactic acid (DA) is the main compound of the aqueous extract of Sm.     

Enhanced thioredoxin,glutathione and Nrf2 antioxidant systems by safflower extract and aceglutamide attenuate cerebral ischaemia/reperfusion injury

A large number of reactive oxygen species (ROS) aggravate cerebral damage after ischaemia/reperfusion (I/R). Glutathione (GSH), thioredoxin (Trx) and nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) represent three major antioxidant systems and play vital roles in affecting each other in eliminating ROS. Identification of drugs targeting triple antioxidant systems simultaneously is vital for inhibiting oxidative damage after cerebral I/R. This study investigated the protective effect of safflower extract and aceglutamide (SAAG) against cerebral I/R injury through modulating multiple antioxidant systems of GSH, Trx and Nrf2 and identified each role of its component acegluatminde (AG) and safflower extract (SA) on these systems. Safflower extract and aceglutamide and its two components decreased neurological deficit scores, infarction rate, apoptosis and oxidative damage after cerebral I/R while enhanced cell viability, decreased reactive oxygen species and nitric oxide level in H₂O₂‐induced PC12 cell model. Importantly, compared to its two components, SAAG demonstrated more effective enhancement of GSH, Nrf2 and Trx systems and a better protection against cerebral I/R injury. The enhanced antioxidant systems prevented ASK1 activation and suppressed subsequent p38 and JNK cascade‐mediated apoptosis. Moreover, inhibition of Trx and Nrf2 systems by auranofin and ML385 abolished SAAG‐mediated protection, respectively. Thus, enhanced triple systems by SAAG played a better protective role than those by SA or AG via inhibition of ASK1 cascades. This research provided evidence for the necessity of combination drugs from the perspective of multiple antioxidant systems. Furthermore, it also offers references for the study of combination drugs and inspires novel treatments for ischaemic stroke.     

CFTR Expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis

Production of hypochlorous acid (HOCl) in neutrophils, a critical oxidant involved in bacterial killing, requires chloride anions. Because the primary defect of cystic fibrosis (CF) is the loss of chloride transport function of the CF transmembrane conductance regulator (CFTR), we hypothesized that CF neutrophils may be deficient in chlorination of bacterial components due to a limited chloride supply to the phagolysosomal compartment. Multiple approaches, including RT-PCR, immunofluorescence staining, and immunoblotting, were used to demonstrate that CFTR is expressed in resting neutrophils at the mRNA and protein levels. Probing fractions of resting neutrophils isolated by Percoll gradient fractionation and free flow electrophoresis for CFTR revealed its presence exclusively in secretory vesicles. The CFTR chloride channel was also detected in phagolysosomes, a special organelle formed after phagocytosis. Interestingly, HL-60 cells, a human promyelocytic leukemia cell line, upregulated CFTR expresssion when induced to differentiate into neutrophils with DMSO, strongly suggesting its potential role in mature neutrophil function. Analyses by gas chromatography and mass spectrometry (GC-MS) revealed that neutrophils from CF patients had a defect in their ability to chlorinate bacterial proteins from Pseudomonas aeruginosa metabolically prelabeled with [(13)C]-l-tyrosine, unveiling defective intraphagolysosomal HOCl production. In contrast, both normal and CF neutrophils exhibited normal extracellular production of HOCl when stimulated with phorbol ester, indicating that CF neutrophils had the normal ability to produce this oxidant in the extracellular medium. This report provides evidence which suggests that CFTR channel expression in neutrophils and its dysfunction affect neutrophil chlorination of phagocytosed bacteria.