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.

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.

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.

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.