Phytochemical and Biological Investigation of Helianthemum nummularium, a High-Altitude Growing Alpine Plant Overrepresented in Ungulates Diets.

Helianthemum nummularium is a European shrub growing at high altitude where it copes with a high level of stress. It was found to be overexpressed in ungulates diets compared to more abundant surrounding plants. These elements combined with the fact that H. nummularium from the Alps has never been investigated prompted us to study the phytochemical composition of its aerial parts. The analysis of the polar extract allowed for the isolation of eight compounds: p-hydroxybenzoic acid, tiliroside, kaempferol, astragalin, quercetin, plantainoside B, quercetin-3-O-glucoside, and quercetin-3-O-glucuronide. We investigated the effect of the polar extract and isolated compounds on nuclear factor erythroid 2-related factor 2 transcription factor, which regulates the expression of a wide variety of cytoprotective genes. We found that the ethanolic extract activates the expression of nuclear factor erythroid 2-related factor 2 in a dose-dependent manner, whereas the pure compounds were much less active. The activation of the nuclear factor erythroid 2-related factor 2 pathway by the plant extract could pave the way for studies to promote healthy aging through protection of cells against oxidative stress. Moreover, the isolated compounds could be investigated alone or in combination in the perspective of making the link between the ungulate's preference for this plant and possible use of it for self-medication.

Phytochemical Combination PB125 Activates the Nrf2 Pathway and Induces Cellular Protection against Oxidative Injury.

Bioactive phytochemicals in Rosmarinus officinalis, Withania somnifera, and Sophora japonica have a long history of human use to promote health. In this study we examined the cellular effects of a combination of extracts from these plant sources based on specified levels of their carnosol/carnosic acid, withaferin A, and luteolin levels, respectively. Individually, these bioactive compounds have previously been shown to activate the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, which binds to the antioxidant response element (ARE) and regulates the expression of a wide variety of cytoprotective genes. We found that combinations of these three plant extracts act synergistically to activate the Nrf2 pathway, and we identified an optimized combination of the three agents which we named PB125 for use as a dietary supplement. Using microarray, quantitative reverse transcription-PCR, and RNA-seq technologies, we examined the gene expression induced by PB125 in HepG2 (hepatocellular carcinoma) cells, including canonical Nrf2-regulated genes, noncanonical Nrf2-regulated genes, and genes which appear to be regulated by non-Nrf2 mechanisms. Ingenuity Pathway Analysis identified Nrf2 as the primary pathway for gene expression changes by PB125. Pretreatment with PB125 protected cultured HepG2 cells against an oxidative stress challenge caused by cumene hydroperoxide exposure, by both cell viability and cell injury measurements. In summary, PB125 is a phytochemical dietary supplement comprised of extracts of three ingredients, Rosmarinus officinalis, Withania somnifera, and Sophora japonica, with specified levels of carnosol/carnosic acid, withaferin A, and luteolin, respectively. Each ingredient contributes to the activation of the Nrf2 pathway in unique ways, which leads to upregulation of cytoprotective genes and protection of cells against oxidative stress and supports the use of PB125 as a dietary supplement to promote healthy aging.

Nrf2 activation: a potential strategy for the prevention of acute mountain sickness.

Reactive oxygen species (ROS) formed during acute high altitude exposure contribute to cerebral vascular leak and development of acute mountain sickness (AMS). Nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2) is a transcription factor that regulates expression of greater than 90% of antioxidant genes, but prophylactic treatment with Nrf2 activators has not yet been tested as an AMS therapy. We hypothesized that prophylactic activation of the antioxidant genome with Nrf2 activators would attenuate high-altitude-induced ROS formation and cerebral vascular leak and that some drugs currently used to treat AMS symptoms have an additional trait of Nrf2 activation. Drugs commonly used to treat AMS were screened with a luciferase reporter cell system for their effectiveness to activate Nrf2, as well as being tested for their ability to decrease high altitude cerebral vascular leak in vivo. Compounds that showed favorable results for Nrf2 activation from our screen and attenuated high altitude cerebral vascular leak in vivo were further tested in brain microvascular endothelial cells (BMECs) to determine if they attenuated hypoxia-induced ROS production and monolayer permeability. Of nine drugs tested, with the exception of dexamethasone, only drugs that showed the ability to activate Nrf2 (Protandim, methazolamide, nifedipine, amlodipine, ambrisentan, and sitaxentan) decreased high-altitude-induced cerebral vascular leak in vivo. In vitro, Nrf2 activation in BMECs before 24h hypoxia exposure attenuated hypoxic-induced hydrogen peroxide production and permeability. Prophylactic Nrf2 activation is effective at reducing brain vascular leak from acute high altitude exposures. Compared to acetazolamide, methazolamide may offer better protection against AMS. Nifedipine, in addition to its known vasodilatory activities in the lung and protection against high altitude pulmonary edema, may provide protection against brain vascular leak as well.

Protandim does not influence alveolar epithelial permeability or intrapulmonary oxidative stress in human subjects with alcohol use disorders.

Alcohol use disorders (AUDs), including alcohol abuse and dependence, have been linked to the development of acute lung injury (ALI). Prior clinical investigations suggested an association between AUDs and abnormal alveolar epithelial permeability mediated through pulmonary oxidative stress that may partially explain this relationship. We sought to determine if correcting pulmonary oxidative stress in the setting of AUDs would normalize alveolar epithelial permeability in a double-blinded, randomized, placebo-controlled trial of Protandim, a nutraceutical reported to enhance antioxidant activity. We randomized 30 otherwise healthy AUD subjects to receive directly observed inpatient oral therapy with either Protandim (1,350 mg/day) or placebo. Subjects underwent bronchoalveolar lavage (BAL) and blood sampling before study drug administration and after 7 days of therapy; all AUD subjects completed the study protocol without adverse events. BAL total protein was measured at each timepoint as an indicator of alveolar epithelial permeability. In subjects with AUDs, before study drug initiation, BAL total protein values were not significantly higher than in 11 concurrently enrolled controls (P = 0.07). Over the 7-day study period, AUD subjects did not exhibit a significant change in BAL total protein, regardless of their randomization to Protandim {n = 14, -2% [intraquartile range (IQR), -56-146%]} or to placebo [n = 16, 77% (IQR -20-290%); P = 0.19]. Additionally, among those with AUDs, no significant changes in BAL oxidative stress indexes, epithelial growth factor, fibroblast growth factor, interleukin-1ß, or interleukin-10 were observed regardless of drug type received. Plasma thiobarbituric acid reactive substances, a marker of lipid peroxidation, decreased significantly over time among AUD subjects randomized to placebo (P < 0.01). These results suggest that Protandim for 7 days in individuals with AUDs who are newly abstinent does not alter alveolar epithelial permeability. However, our work demonstrates the feasibility of safely conducting clinical trials that include serial bronchoscopies in a vulnerable population at risk for acute lung injury.

Oxidative stress in health and disease: the therapeutic potential of Nrf2 activation.

For the past 40 years or so, oxidative stress has been increasingly recognized as a contributing factor in aging and in various forms of pathophysiology generally associated with aging. Our view of oxidative stress has been largely "superoxide-centric", as we focused on the pathological sources of this oxygen-derived free radical and the types of molecular havoc it can wreak, as well as on the protection provided by the antioxidant enzymes, especially the superoxide dismutases, catalases, and glutathione peroxidases. In the last decade our view of oxidative stress has broadened considerably, and it is now often seen as an imbalance that has its origins in our genes, and the ways in which gene expression is regulated. At the center of this new focus is the transcription factor called nuclear factor (erythroid-derived 2)-like 2, or Nrf2. Nrf2 is referred to as the "master regulator" of the antioxidant response, modulating the expression of hundreds of genes, including not only the familiar antioxidant enzymes, but large numbers of genes that control seemingly disparate processes such as immune and inflammatory responses, tissue remodeling and fibrosis, carcinogenesis and metastasis, and even cognitive dysfunction and addictive behavior. Thus, the dysregulation of Nrf2-regulated genes provides a logical explanation for the connections, both direct and indirect, between observable oxidative stress and perhaps 200 human diseases involving these various physiological processes, each reflecting a network involving many gene products. The evolutionary self-association of these many genes under the common control of Nrf2 suggests that the immune and inflammatory systems may present the largest demand for increased antioxidant protection, apart from constitutive oxidative stress resulting from mitochondrial oxygen consumption for metabolic purposes. Gene expression microarray data on human primary vascular endothelial cells and on the SK-N-MC human neuroblastoma-derived cell line have been obtained in response to the dietary supplement Protandim, a potent composition of highly synergistic phytochemical Nrf2 activators. Pathway analysis of results shows significant modulation by Protandim of pathways involving not only antioxidant enzymes, but of those related to colon cancer, cardiovascular disease, and Alzheimer disease.

Sustained lung activity of a novel chimeric protein, SOD2/3, after intratracheal administration.

Delivery of recombinant superoxide dismutase to the lung is limited by its short half-life and poor tissue penetration. We hypothesized that a chimeric protein, SOD2/3, containing the enzymatic domain of manganese superoxide dismutase (SOD2) and the heparan-binding domain of extracellular superoxide dismutase (SOD3), would allow for the delivery of more sustained lung and pulmonary vascular antioxidant activity compared to SOD2. We administered SOD2/3 to rats by intratracheal (i.t.), intraperitoneal (i.p.), or intravenous (i.v.) routes and evaluated the presence, localization, and activity of lung SOD2/3 1 day later using Western blot, immunohistochemistry, and SOD activity gels. The effect of i.t. SOD2/3 on the pulmonary and systemic circulation was studied in vivo in chronically catheterized rats exposed to acute hypoxia. Active SOD2/3 was detected in lung 1 day after i.t. administration but not detected after i.p. or i.v. SOD2/3 administration or i.t. SOD2. The physiologic response to acute hypoxia, vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation, was enhanced in rats treated 1 day earlier with i.t. SOD2/3. These findings indicate that i.t. administration of SOD2/3 effectively delivers sustained enzyme activity to the lung as well as pulmonary circulation and has a longer tissue half-life compared to native SOD2. Further testing in models of chronic lung or pulmonary vascular diseases mediated by excess superoxide should consider the longer tissue half-life of SOD2/3 as well as its potential systemic vascular effects.

The Dietary Supplement Protandim Decreases Plasma Osteopontin and Improves Markers of Oxidative Stress in Muscular Dystrophy Mdx Mice.

Therapeutic options for Duchenne muscular dystrophy (DMD), the most common and lethal neuromuscular disorder in children, remain elusive. Oxidative damage is implicated as a pertinent factor involved in its pathogenesis. Protandim((R)) is an over-the-counter supplement with the ability to induce antioxidant enzymes. In this study we investigated whether Protandim((R)) provided benefit using surrogate markers and functional measures in the dystrophin-deficient (mdx)mouse model of DMD. Male 3-week-old mdx mice were randomized into two treatment groups: control (receiving standard rodent chow) and Protandim((R))-supplemented standard rodent chow. The diets were continued for 6-week and 6-month studies. The endpoints included the oxidative stress marker thiobarbituric acid-reactive substances (TBARS), plasma osteopontin (OPN), plasma paraoxonase (PON1) activity, H&E histology, gadolinium-enhanced magnetic resonance imaging (MRI) of leg muscle and motor functional measurements. The Protandim((R)) chow diet in mdx mice for 6 months was safe and well tolerated. After 6 months of Protandim((R)), a 48% average decrease in plasma TBARS was seen; 0.92 nmol/mg protein in controls versus 0.48 nmol/mg protein in the Protandim((R)) group (p = .006). At 6 months, plasma OPN was decreased by 57% (p = .001) in the Protandim((R))-treated mice. Protandim((R)) increased the plasma antioxidant enzyme PON1 activity by 35% (p = .018). After 6 months, the mdx mice with Protandim((R)) showed 38% less MRI signal abnormality (p = .07) than mice on control diet. In this 6-month mdx mouse study, Protandim((R)) did not significantly alter motor function nor histological criteria.

The induction of human superoxide dismutase and catalase in vivo: a fundamentally new approach to antioxidant therapy.

A composition consisting of extracts of five widely studied medicinal plants (Protandim) was administered to healthy human subjects ranging in age from 20 to 78 years. Individual ingredients were selected on the basis of published findings of induction of superoxide dismutase (SOD) and/or catalase in rodents in vivo, combined with evidence of decreasing lipid peroxidation. Each ingredient was present at a dosage sufficiently low to avoid any accompanying unwanted pharmacological effects. Blood was analyzed before supplementation and after 30 and 120 days of supplementation (675 mg/day). Erythrocytes were assayed for SOD and catalase, and plasma was assayed for lipid peroxidation products as thiobarbituric acid-reacting substances (TBARS), as well as uric acid, C-reactive protein, and cholesterol (total, LDL, and HDL). Before supplementation, TBARS showed a strong age-dependent increase. After 30 days of supplementation, TBARS declined by an average of 40% (p = 0.0001) and the age-dependent increase was eliminated. By 120 days, erythrocyte SOD increased by 30% (p < 0.01) and catalase by 54% (p < 0.002). We conclude that modest induction of the catalytic antioxidants SOD and catalase may be a much more effective approach than supplementation with antioxidants (such as vitamins C and E) that can, at best, stoichiometrically scavenge a very small fraction of total oxidant production.

Stability and conformation of the complexes of riboflavin with aromatic hydroxy compounds in an aqueous medium.

Overall equilibrium constants K' for the formation of molecular complexes of riboflavin with the conjugate forms of different aromatic hydroxy compounds are greater in magnitude than those involving the protonated forms of the hydroxy compounds. There is good agreement between the K(B) values determined by using absorption and emission methods. Based on the magnitude of K(B), it is possible to differentiate between the donor capacities of the hydroxy compounds in the charge transfer complexes with riboflavin. Molecular modeling studies indicate stacking interactions for all the systems in an aqueous medium.

Studies on the riboflavin-resorcinol interaction in an aqueous medium and its pH dependence.

The equilibrium constants, K', for the formation of the molecular association complex between riboflavin and resorcinol in the pH range 6-8 were found to be in the range 5-25 mol(-1)dm(3) by difference absorption spectroscopy. The equilibrium constants from emission spectroscopy were estimated to be in the range 38-55 mol(-1)dm(3) over the same pH range. Both sets of K' show a remarkable dependence on pH. The equilibrium constants K(A) and K(B) describing the interaction of riboflavin with neutral and ionic forms of resorcinol, respectively, obtained by resolving K', shows that K(B) is much greater than K(A). Molecular modeling studies suggest a stacked conformation of the two components in the complexed form. The interaction energies obtained from modeling studies also suggest a stronger interaction between the ionic form of resorcinol and riboflavin.

Oxidative stress in organ preservation: a multifaceted approach to cardioplegia.

Every transplant is a reperfused organ and, therefore, undergoes some degree of oxidative damage. Postischemic reperfusion injury results in non-specific free radical-mediated acute endothelial damage, cell death and organ failure. The endothelium is a key site of injury from reactive oxygen species (ROS), and the endothelial cell dysfunction is central to the pathogenesis of arteriosclerosis. Accelerated arteriosclerosis, secondary to chronic allograft rejection, is a major long-term complication of heart transplantation. Therefore, preservation methods that would decrease injury during reperfusion are very important. We have developed a unique preservation solution, with a multifaceted approach, which best preserves the organ from ROS for an extended period of time before transplantation. The advantages of extending this period of preservation include an expansion of the donor pool, by permitting more distant procurement, the ability to perform detailed tissue typing, therefore, improves histocompatibility match and a reduction in emergency surgery as a result of graft rejection.

Synthesis and anti-inflammatory activity of a chimeric recombinant superoxide dismutase: SOD2/3.

External surfaces of cells are normally protected by extracellular superoxide dismutase, SOD3, which binds to polyanions such as heparan sulfate. We constructed a fusion gene encoding a chimeric SOD consisting of the mature human mitochondrial SOD2 plus the COOH-terminal 26-amino acid heparin-binding "tail" from SOD3. This tail is responsible for the enzyme's affinity for endothelial surfaces. The fusion gene was expressed in Escherichia coli, and the fully active enzyme SOD2/3 was purified. Although native SOD2 has no affinity for heparin, SOD2/3 binds to a heparin-agarose column. In a rat model of acute lung injury induced by intratracheal instillation of IL-1, SOD2/3, SOD2, and denatured SOD2/3 showed 92%, 13.8%, and 0% reduction of lung leak, respectively. Only SOD2/3 prevented neutrophil accumulation. In the carrageenan-induced foot edema model in the rat, SOD2/3 reduced edema by 62% (P < 0.003) at a dose in which native SOD2 produced no significant effect. Thus SOD2/3 appears to have properties as a therapeutic anti-inflammatory agent that are greatly superior to other available forms of the enzyme.

A novel heparin-binding, human chimeric, superoxide dismutase improves myocardial preservation and protects from ischemia-reperfusion injury.

BACKGROUND: The plasma membranes of endothelial cells are sites of physiologic injury caused by superoxide attack, whether the radicals are generated within the cell (i.e., from enzymatic sources such as xanthine oxidase or from ischemically injured mitochondria) or are generated within the interstitial spaces by activated neutrophils or macrophages. An extracellular superoxide dismutase (ECSOD) electrostatically bound to endothelial surfaces partially protects against this oxidative attack. To provide a therapeutic equivalent of this ECSOD activity, we evaluated the product of a fusion gene encoding a chimeric manganese SOD (chimeric-SOD) and the carboxyl-terminal 26-amino acid basic "tail" from ECSOD with high affinity for heparin-like proteoglycans on cell surfaces. METHODS: We tested the chimeric-SOD in isolated rabbit hearts during warm and cold ischemia. RESULTS: When perfused through an isolated rabbit heart, chimeric-SOD bound to endothelial surfaces and was displaced by a bolus dose of heparin. In an established model of no-flow ischemia followed by reperfusion of the isolated rabbit heart, the chimeric-SOD was as protective as native Mn-SOD or Cu,Zn-SOD, but at doses nearly 2 orders of magnitude lower. In a rabbit-heart preservation model, the chimeric-SOD provided better recovery of function after 4 hours of cold ischemia than did University of Wisconsin cardioplegia solution. CONCLUSION: This chimeric-SOD can bind to cell surfaces and may aid in preventing superoxide-mediated endothelial damage and may function as a rational therapeutic agent for treating free-radical-mediated diseases.