Rapid and convenient detection of ascorbic acid: application of fluorescent nitroxide switch

Mesophytic plants possess a set of isoperoxidases with functions known or unknown to us. Usually the peroxidases have their optimum temperature between 25 and 45 degree celsius. Occurance of peroxidase which is thermotolerant is an unusual phenomenon. Aegle marmelos, a plant of family rutaceae, is known in India for its biopharmaceutical significance. Its antioxidant property is due to presence of alkaloids, polyphenolics and flavanoids displaying tissue and stage dependent variation in content and quality. So is the case with enzymatic antioxidants to which belongs, peroxidases. A thermotolerant peroxidase has been isolated and characterized from leaves of Aegle marmelos using ammonium sulphate fractionation and ion exchange and gel permeation column chromatography. This peroxidase is unique in tolerance to temperature as high as 80 °C. The peroxidase would be used to tag secondary antibody for detection of rective antigen using ELISA and would also be used to tag nanoparticles in order to monitor the transformed cells/ tissues.

multiplex gene expression panel to determine nutrigenomic contribution of each bioactive studied. Results: All bioactives up-regulated SOD 2 gene in a dose-dependent manner, while only GABA and oryzanol up-regulated SOD 1 and catalase genes, respectively. Discussion: Apparently, synergism plays a role in nutrigenomic up-regulation of antioxidant genes by GBR bioactives. The implication of our findings is that for half the world population dependent on WR as a staple food, using GBR as a substitute may reduce or prevent the risk of developing oxidative stress-related diseases. Additionally, nutraceuticals may be developed from these bioactives, which could also benefit non-rice eating populations. Keywords: Antioxidant genes, Germinated brown rice, White rice

Oxidative

stress,

doi:10.1016/j.freeradbiomed.2012.08.221

Keywords: Thermotolerant peroxidase, Aegle marmelos doi:10.1016/j.freeradbiomed.2012.08.220 [0468] Nutrigenomic effects of germinated brown rice bioactives on antioxidant genes M.U. Imam*, M. Ismail, A.R. Omar Universiti Putra Malaysia, Malaysia Introduction: White rice (WR) has high glycemic index and therefore could promote oxidative stress, and predispose to diseases like type 2 diabetes mellitus, cardiovascular diseases and other oxidative stressrelated diseases. Germinated brown rice (GBR) on the other hand is known to contain bioactives with high antioxidant potentials. Our preliminiary data showed that GBR improved antioxidant status (total antioxidant status, and hydroxyl radiacal scavenging activities of liver and kidneys) in type 2 diabetic rats through upregulation of antioxidant genes (superoxide dismutase [SOD] and catalase), giving us reason to further study the contributions of different GBR bioactives towards such mechanism. Methods: Bioactives were extracted using different solvents, and quantified on HPLC-DAD (γ-amino butyric acid [GABA], oryzanol and phenolic rich fraction) and GC-MS/MS QqQ (acylated steryl glycoside [ASG]). MTT assays were done on cultured HEP-G2 cells to determine toxicity. Cells were then treated with non-toxic concentrations (50 ppm and 100 ppm) of the bioactives, and SOD 1, SOD 2 and catalase genes studied on a

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[0471] Hepatoprotective and Antioxidant activity of Rhizome of Podophyllum hexandrum against Carbon Tetra Chloride induced Hepatotoxicity in Rats S. Ganie*, B. Zargar, A. Masood, A. Zargar University of Kashmir, India Objective: In the present study n-hexane extract of Podophyllum hexandrum was tested for its possible antioxidant activity in in vitro and in vivo conditions. The in vitro antioxidant activity was evaluated by the ability of the extract to interact with the stable free radical DPPH, Superoxide (O2•−), Hydroxyl (OH•), Hydrogen peroxide (H2O2) radicals and reducing power ability of the extract was also evaluated. Under in vivo conditions the extract was evaluated for its hepatoprotective activity by measuring different biochemical parameters such as serum alanine aminotransaminase, serum aspartate aminotransaminase and serum lactate dehydrogenase and antioxidant enzymes. Methods: Group I (control) was given olive oil, while the rest groups were injected intraperitoneally with a single dose of CCl4 (1 mg/kg) as a 50% (v/v) solution in olive oil. Group III animals received vitamin E at a concentration of 50mg/kg body weight and animals of groups IV, V and VI were given different concentrations of n- hexane extract of Podophyllum hexandrum. Antioxidant status was estimated by determining the activities of antioxidative enzymes, glutathione reductase (GR), glutathione peroxidase (GPX), glutathione-S-

transferase (GST) and superoxide dismutase (SOD); as well as by determining the levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS). Results: Results indicate that hexane extract of P. hexandrum has exhibited the good radical scavenging capacity in neutralization of DPPH, O2•−, OH• and H2O2 radicals in a dose dependent manner. Results from the study showed that n-hexane extract of Podophyllum hexandrum at the doses of (20, 30 and 50 mg/kg-day) produced hepatoprotective effect by decreasing the activity of serum marker enzymes, while it significantly increased the levels of glutathione (GSH), glutathione peroxidase (GPX), glutathione reductase (GR), super oxide dismutase (SOD) and glutathione-S-transferase (GST) in a dose dependant manner. The effect of nhexane extract was comparable to that of standard antioxidant vitamin E. doi:10.1016/j.freeradbiomed.2012.08.222

intensity of Naph-DiPy nitroxide did not enhance in response to various reductants such as glutathione and reactive oxygen species. To confirm the practical usefulness of fluorophore-nitroxide probe, we applied Naph-DiPy nitroxide for the measurement of ascorbic acid in the plasma of osteogenic disorder Shionogi (ODS) rat. Ascorbic acid level decreases in the tissues and plasma of ODS rats, when fed an ascorbic aciddeficient diet. These results suggested that the novel application of fluorophore-nitroxide probe could sensitively and easily detect ascorbic acid and be useful tools for diagnosis in disease state. Keywords: ESR, fluorescence, Nitroxide, ascorbic acid doi:10.1016/j.freeradbiomed.2012.08.223 [0489] Protective Effect Of MitoQ Against Dichlorvos Induced Oxidative Stress And Ensuing Dopaminergic Neuronal Cell Death

[0485] Rapid and convenient detection of ascorbic acid: application of fluorescent nitroxide switch M. Yamato*, Y. Matsuoka, T. Yamasaki, F. Mito, K. Yamada Kyushu University, Japan Ascorbic acid is a small molecule reductant, and has multiple functions in vivo. By reducing ascorbic acid intake, the lack of hydroxylation of prolines and lysines causes a looser triple helix, resulting in scurvy. Ascorbic acid acts as antioxidant in vivo. Ascorbic acid can scavenge superoxide anion radical, hydroxyl radical, and other radicals to prevent oxidative stress such as inflammation. Thus, rapid and convenient detection for ascorbic acid should be useful tools for diagnosis, because ascorbic acid is related to disease state. Nitroxide reduced to the corresponding hydroxylamine by ascorbic acid. Furthermore, a sensitive and novel approach, employing covalent coupling of nitroxide with a fluorophore, leads to intramolecular quenching of fluorescence emission by electron exchange interactions. Here, we show a new fluorophore-nitroxide probe with high sensitivity and selectivity for ascorbic acid, developed by utilizing the chemical properties of 2,6-substituted piperidine nitroxide. We synthesized three derivatives with various reactivates for ascorbic acid. Among them, Naph-DiPy nitroxide rapidly reacted with ascorbic acid and show fluorescence enhancement. However, fluorescence

W.Y. Wani*, A. Sunkaria, D.R. Sharma, K.D. Gill PGIMER, Chandigarh, India Introduction: Parkinson's disease (PD) is a common neurodegenerative disease characterized by loss of nigrostriatal dopamine. Evidences suggest that pesticides are one of the leading candidates of environmental toxins that may contribute to the pathogenesis of PD. We have previously shown that loss of dopamine neurons in dichlorvos exposed animals is due to oxidative damage to mitochondria in rat brains. MitoQ is an antioxidant selectively targeted to mitochondria that protects mitochondria from oxidative damage and has been shown to decrease mitochondrial damage in various animal models of oxidative stress. The present study was designed to test whether MitoQ administration has any protective effect against dichlorvos mediated oxidative stress and ensuing PC12 cell death. Methods: To assess this we investigated the effects of pretreatment of MitoQ on the ROS generation, lipid peroxidation, glutathione content, protein oxidation, and manganese superoxide dismutase (MnSOD) activity and Tyrosine Hydroxylase (TH) levels in PC12 cells treated with Dichlorvos. Results: Pretreatment of MitoQ (1µM) reduced oxidative stress (decreased ROS production and increased MnSOD activity, increased reduced glutathione content) with decreased lipid peroxidation and protein oxidation. Moreover, MitoQ pretreatment resulted in prevention of Dichlorvos induced reduction of Tyrosine hydroxylase and release of cytochrome c (cyt c) from mitochondria. Furthermore, MitoQ pretreatment also prevented the oxidative stress induced downregulation of Lon protease

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