Kerry Bone 2012 MH2251 1 Standard Process sponsors these webinars to provide healthcare information to practitioners and to provide them the opportunity to hear about the views, recommendations and experiences of other practitioners.

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Standard Process does not and cannot accept responsibility for errors or omissions or for any consequences from applications of the information provided and makes no warranty, expressed or implied, with respect to the information provided.Antioxidants:A New Understanding Kerry Bone Co-Founder and Director R&D MediHerb AdjunctAssociate Professor University of New England, Australia Topics Covered Basic redox chemistry and definitions Herbal (phytochemical) antioxidants: important considerations and controversies The essential aspects of the Nrf2/ARE pathway - a new insight into clinically relevant antioxidant protection The clinical relevance of the pathway for health, wellness and normal processes of aging Herbs and phytochemicals shown to act via the Nrf2/ARE pathway Current and novel clinical applications informed by this new understanding 3 Kerry Bone 2012 MH2252 Basic Redox Chemistry and Definitions Redox (short for reduction-oxidation) describes chemical reactions where the oxidation state of component atoms changes With oxidation (one half of the redox reaction) there is a loss of electrons or an addition of oxygen With reduction (the other half of the redox reaction) there is a gain of electrons or an addition of hydrogen In a redox reaction the oxidant (oxidizing agent) is reduced and the reducing agent is oxidized 4 Basic Redox Chemistry and Definitions Hence there is potentially a flow of electrons from the reducing agent to the oxidant A chemical that is good at giving away electrons is a powerful reducing agent Similarly, a chemical that actively seeks electrons is a powerful oxidising agent Because electron flow is often involved, chemicals involved in redox reactions can be placed in an electrochemical series, depending on their redox potentials (measured in volts) The more negative the redox potential, the more powerful the reducing agent 5 Basic Redox Chemistry and Definitions The more positive the potential, the more powerful the oxidant When a chemical is placed in this series, anything with a more negative redox potential can reduce it, anything with a more positive potential can oxidize it Hence, depending on the environment and what it reacts with, an antioxidant (reducing) chemical can become a pro-oxidant (oxidizing agent) Moreover, any chemical entity involved in a redox reaction has an oxidized form (that can act as pro-oxidant) and a reduced form (that can act as an antioxidant) 6 Kerry Bone 2012 MH2253 Basic Redox Chemistry and Definitions In the human body, many key oxidizing agents are based on oxygen and are called reactive oxygen species (ROS) ROS are produced as a normal product of cellular metabolism Many ROS have unpaired electrons that need to find a pair, such chemicals are known as free radicals Free radicals can affect tissue by causing cross linking of molecules and chain reactions 7 The Value of Antioxidants One of the most critical functions of life is the ability to manage oxidative stress1 Oxygen is a two-edged sword: it provides energy for life but the by-products (ROS) are corrosive to tissue, just as oxygen in the atmosphere is corrosive to metal The body is exposed to other pro-oxidants via electromagnetic radiation and the things we ingest or inhale Hence, management of oxidative stress, especially within the cell, is critical to its viability ROS have been implicated in the etiology of a range of health challenges 1Maher J, Yamamoto M. Toxicol Appl Pharmacol 2010; 244(1): 4-15 8 9 Kerry Bone 2012 MH2254 Controversies Over Antioxidants The oxidative stress theory of aging (and hence the value of consuming antioxidant supplements) has been challenged by many researchers, even former advocates (see later in slide 13) Antioxidants might become pro-oxidants in the right environment: examples include vitamin C and the negative clinical results for isolated beta-carotene in smokers The discovery of ROS signalling: demonstrated in a variety of physiological processes including glucose-stimulated insulin secretion and induction of host defenses1 1Zhang Q, Pi J, Woods CG et al. Toxicol Appl Pharmacol 2010; 244(1): 84-97 10 Herbal Antioxidants are Misunderstood A number of methods are used to assess the antioxidant activity of plant-based foods and therapeutic agents These include ORAC (oxygen radical absorbance capacity), TRAP (total radical-trapping antioxidant parameter) and TEAC (trolox equivalent antioxidant capacity) However, they are all test tube methods that probably have little bearing on what actually happens in the body after the plant is ingested 11 Herbal Antioxidants are Misunderstood Specifically, it has been shown that often only a small fraction of the antioxidant phytochemicals that contribute to ORAC etc are absorbed as such Or what is absorbed exerts relatively negligible activity directly quenching ROS in body fluids This is supported by a lack of co-ordination between ORAC levels and the ability to control oxidative stress in the body In any case, bodily fluids already contain significant levels of an active antioxidant molecule: uric acid Finley JW, Kong AN, Hintze KJ et al. J Agric Food Chem 2011; 59(13): 6837-6846 Niki E. Curr Pharm Biotechnol 2010; 11(8): 801-809 Cheli F, Baldi A. J Food Sci 2011; 76(9): R197-R20512 Kerry Bone 2012 MH2255 ORAC Values 13 The Oxidative Stress Theory of Aging This was first proposed by Harman in 1956: ROS inflict indiscriminateoxidate damage to cells that cannot be completely neutralized by antioxidants and/or repaired Damage then accumulates with time aging Despite the intuitive logic and vast support for this theory, a causal link between oxidative stress and the rate of aging has not been clearly established1 Many workers now question its validity 1Lewis KN, Mele J, Hayes JD et al. Integr Comp Biol 2010; 50(5): 829-843 14 A New Perspective However, a common denominator exists in several studies: cells from long-living animals show greater levels of cytoprotection They are more resistant to oxidative and chemical insults This strong relationship between species longevity and cellular resistance to oxidative insults has been reported in a wide variety of species across the animal kingdom (see later in slide 23) 15 Kerry Bone 2012 MH2256 16 Important Cellular Protector Pathways The Nrf2/ARE Pathway Otherwise known as the multiorgan protector pathway, this is a basic cellular survival mechanism A primordial pathway, fundamental to all animals cells, it describes a targeted approach to antioxidant protection in the cell The Nrf2/ARE pathway is a dynamic response induced by oxidative/chemical stress on the cell that is fundamentally new and novel It is a switch-on, switch-off mechanism, and most of the priming agents studied to date that facilitate its responsivity are natural plant chemicals 17 The Nrf2/ARE Pathway Phytochemicals exerting a priming effect on the NRf2/ARE pathway are hormetic and represent an indirect way to safely provide antioxidant protection for the cell And the priming of this pathway brings much more than antioxidant effects 18 Kerry Bone 2012 MH2257 The Nrf2/ARE Pathway 19 The Nrf2/ARE Pathway 20 Functions of the Nrf2/ARE Pathway As noted, Nrf2 is expressed in all tissues, but levels vary, with higher levels in key detoxifying organs: the liver and kidneys Activation of Nrf2 activates more than 200 genes Nrf2 activation enhances DNA repair, heme metabolism, efflux transport of toxins and glutathione synthesis It activates detoxification, stabilizes proteins, strengthens cellular integrity and moderates the inflammatory response process 21 Kerry Bone 2012 MH2258 Functions of the Nrf2/ARE Pathway Key antioxidant enzymes are induced, including catalase, superoxide dismutase (SOD), thioredoxin, peroxiredoxin, sulfiredoxin, ferritin, metallothionein and haem oxygenase 1 (HO-1) Key phase II enzymes are also induced, including glutathione S-transferases (GSTs) and NAD(P)H: quinone oxidoreductase 1 (NQO1) These functions likely represent only a fraction of the true function of Nrf2 activity, some of which are only beginning to be understood Maher J, Yamamoto M. Toxicol Appl Pharmacol 2010; 244(1): 4-15 Lewis KN, Mele J, Hayes JD et al. Integr Comp Biol 2010; 50(5): 829-843 Itoh K, Mimura J, Yamamoto M. Antioxid Redox Signal 2010; 13(11): 1665-1678 22 The Nrf2/ARE Pathway in Health and Wellness Current research has identified several important involvements of the Nrf2/ARE pathway in supporting and maintaining health: healthy aging and longevity benefits in health challenges involving natural oxidative stressand normal inflammation potential benefit in health challenges resulting from accumulated environmental challenges 23 The Nrf2/ARE Pathway and Longevity 24 Lewis KN, Mele J, Hayes JD et al. Integr Comp Biol 2010; 50(5): 829-843 Kerry Bone 2012 MH2259 The Nrf2/ARE Pathway and Neuroprotection Beneficial effects of Nrf2/ARE priming/activation have been suggested in neurological health1,2,3,4 HO-1 is a known neuroprotective heat shock protein/enzyme that, for example, supports brain function5 25 The Nrf2/ARE Pathway and Neuroprotection References for previous slide 1Negi G, Kumar A, Joshi RP et al. Oxidative stress and Nrf2 in the pathophysiology of diabetic neuropathy: old perspective with a new angle. Biochem Biophys Res Commun 2011; 408(1): 1-5 2Calkins MJ, Johnson DA, Townsend JA et al. The Nrf2/ARE pathway as a potential therapeutic target in neurodegenerative disease. Antioxid Redox Signal 2009; 11(3): 497-508 3Cuadrado A, Moreno-Murciano P, Pedraza-Chaverri J. The transcription factor Nrf2 as a new therapeutic target in Parkinson's disease. Expert Opin Ther Targets 2009; 13(3): 319-329 4Plafker SM. Oxidative stress and the ubiquitin proteolytic system in age-related macular degeneration. Adv Exp Med Biol 2010; 664: 447-456 5Jazwa A, Cuadrado A. Targeting heme oxygenase-1 for neuroprotection and neuroinflammation in neurodegenerative diseases. Curr Drug Targets 2010; 11(12): 1517-1531 26 The Nrf2/ARE Pathway and Brain Health In one study, cells from biopsy of the olfactory mucosa had lower glutathione levels and detoxifying capacity in patients with brain challenges compared with those from healthy control donors These cells from the challenged patients were also in a state of oxidative stress due to higher levels of hydrogen peroxide Significantly, activation of the Nrf2/ARE pathway restored those defective cells to normal functioningCook AL et al. PLoS One 2011; 6(7): e21907 27 Kerry Bone 2012 MH22510 NRF2/ARE and Other Body Systems Beneficial effects of Nrf2/ARE priming have also been suggested for: lung support cardiovascular support immune system support blood glucose support Singh S, Vrishni S, Singh BK et al. Nrf2-ARE stress response mechanism: a control point in oxidative stress-mediated dysfunctions and chronic inflammatory diseases. Free Radic Res 2010; 44(11): 1267-1288 28 Key Herbs/Phytochemicals with Nrf2/ARE Activity EGCG from Green Tea is also very active (in vitro and in vivo - 20 mg/kg, oral) Curcumin from Turmeric is positive in many studies (in vitro and in vivo, oral) Resveratrol shows marked in vitro and in vivo activities and was also positive in an human trial (see later) Surh YJ, Kundu JK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med 2008; 74(13): 1526-1539 Hur W, Gray NS. Small molecule modulators of antioxidant response pathway. Curr Opin Chem Biol 2011; 15(1): 162-173 29 Key Herbs/Phytochemicals with Nrf2/ARE Activity Carnosol from Rosemary is another well-studied phytochemical Various phytochemicals from Garlic are active in vitro Several flavonoids exhibit in vitro activity, but this may not translate to clinical relevance because of their low oral bioavailability Surh YJ, Kundu JK, Na HK. Nrf2 as a master redox switch in turning on the cellular signaling involved in the induction of cytoprotective genes by some chemopreventive phytochemicals. Planta Med 2008; 74(13): 1526-1539 Hur W, Gray NS. Small molecule modulators of antioxidant response pathway. Curr Opin Chem Biol 2011; 15(1): 162-173 30 Kerry Bone 2012 MH22511 Rosemary Rosemary contains the antioxidant molecules carnosol and carnosic acid Both of these are now understood to be potent primers of the Nrf2/ARE pathway For example both compounds have demonstrated potential neuroprotective activity mediated by this pathway, which reflects on its traditional use for memory1,2 Recently a single 750 mg dose of Rosemary improved speed of memory and alertness in healthy older adults1 1Kosaka K et al. J Biochem 2010; 147(1): 73-81 2Martin D et al. Biol Chem 2004; 279(10): 8919-8929 3Pengelly A et al. J Med Food 2012; 15(1): 10-17 31 32 Turmeric/Curcumin Multiple in vivo studies have demonstrated the activities of curcumin and Turmeric1 The Nrf2/ARE pathway is thought to be an important basis for these effects For example, the epigenetic silencing of Nrf2 during the progression of prostate challenge in a mouse model was reversed by curcumin2 1Aggarwal BB, Surh YJ, Shisodia S (eds). Adv Exptl Med Biol2007; 595;1-489 2Khor TO, Huang Y, Wu TY et al. Biochem Pharmacol 2011; 82(9): 1073-1078 33 Kerry Bone 2012 MH22512 Turmeric/Curcumin In other relevant models, chemically induced oxidative stress was reduced by curcumin in vivo in parallel with Nrf2/ARE upregulation1 Brain and liver were supported in vivo by curcumin through Nrf2-mediated induction of HO-12,3 Dietary curcumin led to increased Nrf2 protein levels and enhanced ARE binding in liver and lungs of mice4 1Charoensuk L et al. Int J Parasitol 2011; 41(6): 615-626 2Yang C et al. Brain Res 2009; 1282: 133-141 3Farombi EO et al. Food Chem Toxicol 2008; 46(4): 1279-1287 4Garg R et al. Carcinogenesis 2008; 29(5): 1022-1032 34 35 36 Kerry Bone 2012 MH22513 Resveratrol Resveratrol is a highly active primer of the Nrf2/ARE pathway Favorable Nrf2-mediated activity has been demonstrated in many body systems including the endocrine, cardiovascular and nervous systems For example, the endothelial (circulatory) protective effects of resveratrol against a high-fat diet were largely diminished in Nrf2 knockout mice1 Hask G, Pacher P. Am J Physiol Heart Circ Physiol 2010; 299(1): H10-H12 37 Resveratrol In a recent double blind, randomized, crossover study, 10 normal, healthy men and women were given a high-fat, high-carbohydrate (HFHC) meal (930 kcal) either with a placebo or a product containing 100 mg of resveratrol from Polygonum cuspidatum plus 75 mg of total polyphenols from a grape extract1 Profound benefits were observed 1Ghanim H, Sia CL, Korzeniewski K et al. J Clin Endocrinol Metab 2011; 96(5): 1409-1414 38 Resveratrol DNA binding activity of Nrf2 in white cells was significantly increased by 15039% over baseline (p