Biological potential of an ethanolic extract from “Mela Rosa Marchigiana” pulp callus culture

The biological effects of the ethanolic extract from Mela Rosa Marchigiana pulp callus were investigated. In terms of the antioxidant activity, the extract exhibited free radical scavenging activity of 67% and 39% using the DPPH assay and ABTS assay respectively. Furthermore, it reduced the ROS production in the keratinocyte cell model of H2O2 induced oxidative stress. The genoprotective effect was evaluated using the DNA nicking assay, which revealed significant protection up to 70%. The anti-inflammatory response was detected at 0.5 mg/ml through the release of nitric oxide using bacterial LPS and RAW 264.7 cells. Finally, preliminary studies on keratinocytes suggested a possible positive effect of the extract on mitochondrial biogenesis and wound healing. The obtained results encourage further studies to deep the biological effects of this callus with the future objective to propose a product for nutraceutical, cosmetic and food-tech industries, as well as an alternative to normal ways of chemical synthesis

Cydonia oblonga Mill. Pulp Callus Inhibits Oxidative Stress and Inflammation in Injured Cells

The pharmacological activity of a callus extract from the pulp of Cydonia oblonga Mill., also known as quince, was investigated in murine macrophage (RAW 264.7) and human keratinocyte (HaCaT) cell lines. In particular, the anti-inflammatory activity of C. oblonga Mill. pulp callus extract was assessed in lipopolysaccharides (LPS)-treated RAW264.7 by the Griess test and in LPS-treated HaCaT human keratinocytes by examining the expression of genes involved in the inflammatory process, including nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1 (IL-1 ), nuclear factor-kappa-B inhibitor alfa (ikB ), and intercellular adhesion molecule (ICAM). The antioxidant activity was evaluated by quantizing the reactive oxygen species (ROS) production in the hydrogen peroxide and tert-butyl hydroperoxide-injured HaCaT cell line. The obtained results indicate that C. oblonga callus from fruit pulp extract has anti-inflammatory and antioxidant activities, suggesting its possible application in delaying and preventing acute or chronic diseases associated with aging or in the treatment of wound dressing

Influenza Virus Down-Modulates G6PD Expression and Activity to Induce Oxidative Stress and Promote Its Replication

none10no: Influenza virus infection induces oxidative stress in host cells by decreasing the intracellular content of glutathione (GSH) and increasing reactive oxygen species (ROS) level. Glucose-6-phosphate dehydrogenase (G6PD) is responsible for the production of reducing equivalents of nicotinamide adenine dinucleotide phosphate (NADPH) that is used to regenerate the reduced form of GSH, thus restoring redox homeostasis. Cells deficient in G6PD display elevated levels of ROS and an increased susceptibility to viral infection, although the consequences of G6PD modulation during viral infection remain to be elucidated. In this study, we demonstrated that influenza virus infection decreases G6PD expression and activity, resulting in an increase in oxidative stress and virus replication. Moreover, the down regulation of G6PD correlated with a decrease in the expression of nuclear factor erythroid 2-related factor 2 (NRF2), a key transcription factor that regulates the expression of the antioxidant response gene network. Also down-regulated in influenza virus infected cells was sirtuin 2 (SIRT2), a NADPH-dependent deacetylase involved in the regulation of G6PD activity. Acetylation of G6PD increased during influenza virus infection in a manner that was strictly dependent on SIRT2 expression. Furthermore, the use of a pharmacological activator of SIRT2 rescued GSH production and NRF2 expression, leading to decreased influenza virus replication. Overall, these data identify a novel strategy used by influenza virus to induce oxidative stress and to favor its replication in host cells. These observations furthermore suggest that manipulation of metabolic and oxidative stress pathways could define new therapeutic strategies to interfere with influenza virus infection.openDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, LuciaDe Angelis, Marta; Amatore, Donatella; Checconi, Paola; Zevini, Alessandra; Fraternale, Alessandra; Magnani, Mauro; Hiscott, John; De Chiara, Giovanna; Palamara, Anna Teresa; Nencioni, Luci

"Shock and kill" effects of class I-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoximine in cell line models for HIV-1 quiescence

Latently infected, resting memory CD4+ T cells and macrophages represent a major obstacle to the eradication of HIV-1. For this purpose, "shock and kill" strategies have been proposed (activation of HIV-1 followed by stimuli leading to cell death). Histone deacetylase inhibitors (HDACIs) induce HIV-1 activation from quiescence, yet class/isoform-selective HDACIs are needed to specifically target HIV-1 latency. We tested 32 small molecule HDACIs for their ability to induce HIV-1 activation in the ACH-2 and U1 cell line models. In general, potent activators of HIV-1 replication were found among non-class selective and class I-selective HDACIs. However, class I selectivity did not reduce the toxicity of most of the molecules for uninfected cells, which is a major concern for possible HDACI-based therapies. To overcome this problem, complementary strategies using lower HDACI concentrations have been explored. We added to class I HDACIs the glutathione-synthesis inhibitor buthionine sulfoximine (BSO), in an attempt to create an intracellular environment that would facilitate HIV-1 activation. The basis for this strategy was that HIV-1 replication decreases the intracellular levels of reduced glutathione, creating a pro-oxidant environment which in turn stimulates HIV-1 transcription. We found that BSO increased the ability of class I HDACIs to activate HIV-1. This interaction allowed the use of both types of drugs at concentrations that were non-toxic for uninfected cells, whereas the infected cell cultures succumbed more readily to the drug combination. These effects were associated with BSO-induced recruitment of HDACI-insensitive cells into the responding cell population, as shown in Jurkat cell models for HIV-1 quiescence. The results of the present study may contribute to the future design of class I HDACIs for treating HIV-1. Moreover, the combined effects of class I-selective HDACIs and the glutathione synthesis inhibitor BSO suggest the existence of an Achilles' heel that could be manipulated in order to facilitate the "kill" phase of experimental HIV-1 eradication strategies

Modulation of the biosynthesis of oxyprenylated coumarins in calli from Ferulago campestris elicited by ferulic acid

Previous and recent literature acquisitions suggested that cultured calli are an efficient and meaningful model to investigate the extent and fate of prenylation of phenylpropanoid cores in plants belonging to the Rutaceae and Apiaceae families upon administration of putative biosynthetic precursors. To this concern, in the present manuscript, we investigated the effect of supplementation of ferulic acid and umbelliferone on the biosynthesis of their oxyprenylated counterparts in Ferulago campestris (Besser) Grecescu (Fam. Apiaceae) cultured calli. Dried plant biomass material has been extracted by an ultrasoundassisted extraction with EtOH. O-prenyl secondary metabolites, namely 3,3-dimethylallyloxy, geranyloxy, and farnesyloxy derivatives of both ferulic acid and umbelliferone, were identified and quantified by HPLC/DAD analyses. Supplementation with ferulic acid was the only treatment providing appreciable results. Quite surprisingly, its addition to cultured calli did not affect the formation of its oxyprenylated phytochemicals but boosted the biosynthesis of umbelliferone and its farnesyloxyderivative umbrelliprenin. The findings reported herein enforce and underline the role of ferulic acid as an elicitor of selected classes of secondary metabolites in apiaceous species, as recently observed. In addition to these results, a novel hitherto unknown metabolite from F. campestris, namely 7-[[(2E)-7-hydroxy-3,7-dimethyl-2-octen-1-yl]oxy]-2H-1-benzopyran- 2-one, was characterized by NMR and LC–MS analyses

High production of secondary metabolites and biological activities of Cydonia oblonga Mill. pulp fruit callus

This study has developed an innovative method for the production of secondary metabolites starting from Cydonia oblonga Mill (quince) pulp callus culture. The qualitative and quantitative content of phenolic and triterpenic acids of quince callus extract were elucidated by GC–MS, GC, and HPLC-DAD-ESI-MSn. The callus extract was rich of 5-O-caffeoylquinic acid (5-CQA), 5-p-coumaroylquinic acid (5-p-CoQA) and maslinic and corosolic acid. Quince callus extract’s radical scavenging and antioxidant activity were evaluated by 2,2- diphenyl-1-picrylhydrazyl, 2,2,-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), and Oxygen Radical Absorbance Capacity methods. The genoprotection was evaluated by gel electrophoresis analysis and quantitative Real-Time PCR. In addition to the good antioxidant activity the quince callus extract is a strong inhibitor of α-glucosidase (IC50 of 0.25 ± 0.02 mg dw/mL) and lipase (IC50 of 1.99 ± 0.005 mg dw/mL), but mild inhibitor of α-amylase. Therefore, this work would be significant for the future development of a nutraceutical approach to the management of hyperglycemia and dyslipidemia

Validation of a Reversed-Phase High Performance Liquid Chromatography method for the simultaneous analysis of cysteine and reduced glutathione in mouse organs

A depletion of reduced glutathione (GSH) has been observed in pathological conditions and in aging. Measuring GSH in tissues using mouse models is an excellent way to assess GSH depletion and the potential therapeutic efficacy of drugs used to maintain and/or restore cellular redox potential. A high performance liquid chromatography (HPLC) method for the simultaneous determination of GSH and cysteine (Cys) in mouse organs was validated according to USA and European standards. The method was based on separation coupled with ultraviolet detection and precolumn derivatization with 5,5 -dithiobis-(2-nitrobenzoic acid) (DTNB). The required validation parameters, that are, selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, and stability, were studied for spleen, lymph nodes, pancreas, and brain. The results showed that the lower limits of quantification were 0.313 M and 1.25 M for Cys and GSH, respectively. Intraday and interday precisions were less than 11% and 14%, respectively, for both compounds. The mean extraction recoveries of Cys and GSH from all organs were more than 93% and 86%, respectively. Moreover, the stability of both analytes during sample preparation and storage was demonstrated. The method was accurate, reliable, consistent, and reproducible and it was useful to determine Cys and GSH in the organs of different mouse strains

GSH and analogs in antiviral therapy

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment. GSH is the most powerful intracellular antioxidant and plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione-S-transferases (GST) and glutathione peroxidases (GPx). As a consequence, the ratio of reduced and oxidized glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. A deficiency in GSH puts the cell at risk for oxidative damage. An imbalance in GSH is observed in a wide range of pathologies, such as cancer, neurodegenerative diseases, cystic fibrosis (CF), several viral infections including HIV-1, as well as in aging. Several reports have provided evidence for the use of GSH and molecules able to replenish intracellular GSH levels in antiviral therapy. This non-conventional role of GSH and its analogs as antiviral drugs is discussed in this chapter. (C) 2008 Elsevier Ltd. All rights reserved