Glucoraphanin - an Indirect Antioxidant Found Naturally in Broccoli That Supports Cell Integrity and Protects Against Free Radical Damage by Jeremy Bartos, Ph.D

GENERAL INFORMATION

Glucoraphanin - An Indirect Antioxidant Found Naturally In Broccoli That Supports Cell Integrity And Protects Against Free Radical Damage By Jeremy Bartos, Ph.D. Scientific & Regulatory Affairs Manager Glanbia Nutritionals

Glanbia Nutritionals | truebroc® White Paper | June 2015

introduction Today’s health-conscious consumers are more informed and want to WHAT MAKES TRUEBROCTM A PREMIER understand what they are eating as well as the benefits of the products ANTIOXIDANT they consume. They seek natural ingredients that have no additives or preservatives and are botanical or organic in nature. Currently, there are a large volume of products on the market that have an antioxidant health > truebrocTM boosts Phase II Enzymes, enhancing the claim. In 2015 alone almost 1000 new products with an antioxidant body’s own removal of free radicals and overall position have been released1, but how well do they really work? Recent detoxification of cellsNon-burning and non-irritating scientific research on broccoli has revealed a “next generation” antioxidant to the stomach that will potentially change the way we choose and utilize our ingested antioxidants. truebrocTM glucoraphanin, a naturally derived phytonutrient extracted from broccoli seeds, is a rechargeable antioxidant that works > Standardized to 13% glucoraphanin – highest with the body’s own cellular protection system. Currently, the most widely concentration available accepted source of ingested antioxidants are short-term “one and done” antioxidants, such as Vitamins C and E and polyphenols such as > Made from selected natural broccoli seeds grown resveratrol (grapes and wine) and EGCG (green tea) that last for only a in California and water extracted in Canada short time in the body. The effects of truebrocTM have the benefit of lasting several days in the body – long after the “one and done” antioxidants have • Complete traceability from field to product dissipated. truebrocTM glucoraphanin, the next generation of antioxidants, • Produced under cGMP activates a sustainable antioxidant system within the body that is > Patent Protected – source and process rechargeable and meets the demands of today’s health conscious consumers who are looking for “clean label” options with clearly stated benefits. > Self-Affirmed GRAS

PRODUCTS WITH ANTIOXIDANT > Recommended dosage: CLAIMS ON THE RISE • Supplements – 30mg of glucoraphanin (230mg of truebrocTM) • Foods – 10-15mg of glucoraphanin (77- 115mg of truebrocTM)

> Applications: • Tablets • Capsules • Powders • Functional Beverages • Functional Foods

Glanbia Nutritionals | truebroc® White Paper | June 2015 These statements have not been evaluated by the Food and Drug Administration. This product is not intended to diagnose, treat, cure or prevent disease. 1

THE IMPORTANCE OF ANTIOXIDANTS Everyone has heard about antioxidants and knows that they are good for them, but does everyone really know how antioxidants work in our bodies? The main purpose of antioxidants is to protect the cells in the body from damage caused by oxidative stress, namely free radicals, which are unstable compounds missing an electron (a negative charge) on their outside layer2. Free radicals are both created as a byproduct of normal cellular respiration as well as environmental byproducts of pollution, sunlight, etc. Because of their instability they “steal” electrons from any source they can find in the body to help stabilize them. The most readily available source of electrons in the body is the phospholipid bilayer that surrounds each cell. Over time, the chronic exposure of the cell walls to free radicals will break down the walls, which can have a variety of negative effects, both acute and chronic. In some cases it is a major cause of a disease, such as seen in Alzheimer’s3. In others, it simply makes an already existing condition worse, as in asthma or Rheumatoid Arthritis4,5,6,7.

By definition, antioxidants are electron donors; they act as sacrificial lambs by offering free radicals another source of electrons besides the cell wall. They can give their electrons to free radicals, neutralizing them and thereby preventing them from stealing any more electrons and doing further damage. The antioxidants can thus help prevent or minimize the build-up of damage over time. Unfortunately, free radical damage is also closely linked with inflammation because the body recognizes the oxidative damage as a threat8. To answer this, the body sends out an army of soldiers in the form of immune cells. These immune “soldiers” fight against anything they perceive to be foreign and once they are done, like casualties in any battlefield, there will be a mass of dead cells. This in itself sends signals for an influx of more immune cell soldiers. What results is a cycle of oxidation - damage – immune cells - more damage. Increasing antioxidant potential in the body provides a solution by helping to prevent a lot of the oxidative damage in the first place and thus also helps minimize localized inflammation, for example in the muscles after rigorous exercise9, 10. Without antioxidants, our cells could be easily damaged by reactive oxygen species, DNA damaging electrophiles, inflammation and radiation.

The body has several ways of removing free radicals before they can cause damage. The method best known to consumers is to ingest antioxidants through food or supplements, such as Vitamin C and polyphenols11,12. Slightly less known are native antioxidants and enzymes produced by the body, such as glutathione and superoxide dismutase13,14,15. And finally there is the Phase II Enzyme System; enzymes that act to increase the body’s native antioxidant and detoxifying pathways. The Phase II Enzyme System is activated by Nrf2, a transcription factor that, when induced, increases the production of the specific antioxidant enzymes16,17,18.

LONG-LASTING ANTIOXIDANT EFFECTS Most dietary antioxidants, such as vitamins and polyphenols, act by donating electrons to free radicals. Once they do this, their antioxidant function is depleted: they work in a “one-and- done” fashion. The production of free radicals continues and if the supply of one-and-done antioxidants runs out then those free radicals will start stealing again, which can lead to oxidative damage. Figure 1 demonstrates the “one and done” effect of various amounts of ingested Vitamin C; unless the body gets constant replenishment (as often as 4 to 6 times a day) of these ingested antioxidants, the antioxidant protection quickly wanes in a matter of hours19.

Native antioxidants such as glutathione and superoxide dismutase are “rechargeable” antioxidants; once they have donated electrons they can be “recharged” by the members of Phase II Enzyme system, thus they can be reused over and over again20,21. One way to ensure this occurs is by activating the Phase II Enzyme system; without it, native antioxidants are also just “one and done”. The major regulator of the genes that code the Phase II Enzymes is nuclear factor erythroid 2-related factor 2 (Nrf2), which binds the antioxidant response element (ARE) region of the gene promoter. This in turn leads to the expression of antioxidant and Phase II Enzymes, demonstrating that Nrf2 is the key to start this Phase II Enzyme system22. Therefore, compounds that can activate Nrf2 can indirectly activate the entire Phase II Enzyme system23. Once activated, the Phase II Enzymes can remain operational for as long as 72 hours, thereby ensuring long-lasting systemic antioxidant effects24. In addition to Phase II Enzyme regulation, Nrf2-ARE binding regulates the expression of more than 100 other genes involved in cellular antioxidant and anti-inflammatory defense such as heat shock proteins and ferritin, pro- and anti-inflammatory enzymes such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1)25,26. It has been shown that down- regulation of Nrf2 can have a variety of negative effects, from increased oxidative stress load and environmental toxin levels to localized inflammation and aberrant mitochondrial biogenesis27,28. Keeping this important pathway up and running is critical to maintaining healthy cellular processes and staving off chronic maladies.

DISCOVERY: A NEW ANTIOXIDANT COMPOUND FOUND IN BROCCOLI

In the early 1990’s, scientists at Johns Hopkins University identified a compound derived from broccoli called sulforaphane29,30. Sulforaphane and its precursor glucoraphanin (sulforaphane glucosinolate) act as part of the plant’s defense system; in fact, sulforaphane is responsible for the characteristic sulfur smell/taste of broccoli. Glucoraphanin belongs to a category of compounds called glucosinolates, which are naturally found in cruciferous vegetables. It is converted into the isothiocyanate sulforaphane by an enzyme found in broccoli called myrosinase or in the body’s gut microflora31. In the broccoli plant, myrosinase and glucoraphanin are kept in different cellular compartments. When the plant is chewed or cut the myrosinase and glucoraphanin can interact, resulting in the conversion of glucoraphanin into sulforaphane. It is the sulforaphane that has the characteristic bitter broccoli taste designed to discourage animals from eating the plant! In the course of their experiments, the JHU scientists discovered that sulforaphane was one of the most potent activators of Nrf2 found in nature29. This led to a flurry of publications demonstrating the effect that sulforaphane, glucoraphanin, and broccoli extracts have on the activation and expression of specific Phase II Enzymes32. Figure 2 demonstrates the effect of sulforaphane on the activation of several of these enzymes24. Quinone Reductase (QR) helps to reduce electrophilic quinones, an important step in cellular detoxification. Glutathione Reductase (GR) catalyzes the reduction of glutathione disulfide (uncharged) to glutathione (charged). Glucose-6- Phosphate Dehydrogenase (G6PD) helps maintain the cellular level of NADPH which in turn maintains the levels of glutathione. Glutathione (GSH) itself is an indirect target that also increases due to increased activity of Glutathione Reductase.

SULFORAPHANE CLINICAL STUDIES SHOW POSITIVE RESULTS

Recent human studies on sulforaphane and broccoli products have also shown positive results, similar to those seen in the animal trials. A 17 subject randomized, crossover dietary trial was set up to examine the effect of standard broccoli (S) and high-glucoraphanin (HG) broccoli (broccoli with three times more glucoraphanin than standard broccoli) as a single meal on the expression of key Phase II Enzymes, compared with water (w)33. Thioredoxin reductase 1 (Tr1) is an enzyme critical to the thioredoxin pathway that “recharges” thioredoxin, an antioxidant similar to glutathione that acts as an electron donor to peroxidases and ribonucleotide reductase to help reduce free radical damage. Glutamate-cysteine ligase modifier subunit (GCLM) is a subunit of gamma-glutamylcysteine synthetase, the first rate-limiting enzyme of glutathione synthesis. As seen in Figure 3, high-glucoraphanin broccoli showed a >2x induction of these key antioxidant Phase II Enzymes over standard broccoli controls and a >20 fold induction over water33.

In another human crossover study, 20 subjects were given a 4 week intervention of either 2 cups broccoli (~218 grams; BV) or a supplement containing fiber and antioxidant micronutrients found in broccoli such as 60 mg Vitamin C and 5000 IU Vitamin A (M+F)34. In this study, they measured changes in F2-isoprostanes (F2iP), which are biomarkers that are becoming widely recognized as a highly reliable index of body-wide oxidative stress and are easily measurable in urine samples. It was found that consuming 218 grams of broccoli/day decreased overall oxidative stress markers by an average of 22%, while the antioxidant vitamins showed no change in isoprostane levels, demonstrating that the glucoraphanin is the key driver for the effects seen (Figure 4).

In a human dose-escalation placebo-controlled trial, 65 subjects were spread out over 5 groups and given different amounts of broccoli sprout homogenate; a placebo, 50 g broccoli sprout homogenate (BSH), 100 g BSH, 150 g BSH, 200 g BSH35. The aim of this study was to determine if the sulforaphane from broccoli stimulates the production and activation of Phase II Enzymes in a dose dependent manner. In this case, they measured changes from baseline in the expression of NADPH quinone oxidoreductase (NOQ1), which reduces hydrogen peroxide free radicals (a toxic byproduct of cellular respiration), and glutathione S transferase P1 (GSTP1), which functions in removal of free radicals and acts in concert with tumor suppressing protein p53. As highlighted in the circles in Figure 5, it was shown that sulforaphane does indeed increase expression of these Phase II Enzymes in a dose dependent manner, as people who consumed lower amounts of BSH per serving showed lower expression than those people who consumed higher amounts of BSH per serving. Therefore, the more sulforaphane you take, the better the response!

THE ALTERNATIVE TO EATING LARGE AMOUNTS OF BROCCOLI

Who wants to eat 200 grams of broccoli sprout homogenate? Just because broccoli is good for you doesn’t mean people want to eat it, especially at levels high enough to have a meaningful effect on their health. In fact, in an online omnibus survey of 1,012 nationally representative U.S. adults ages 18+ conducted in March 2015, nearly 1 in 3 (32%) Americans have found a reason to avoid eating more broccoli36. Furthermore, nearly 2 in 5 (39%) wish they could get the nutritional benefits from broccoli without actually having to eat it! Therefore, this shows that there is a large group of people who are looking for an easier way to get the antioxidant boosting benefits of broccoli.

WHY TRUEBROCTM IS THE ANSWER

Enter truebrocTM, a patented and highly concentrated broccoli seed extract standardized to glucoraphanin, born from the same Johns Hopkins University scientists who first identified the antioxidant benefits of sulforaphane from broccoli. And this is important because broccoli is variable. The levels of the actives in broccoli vary wildly due to genetic variations, the location where it’s grown, and on the part of the plant consumed (florets, stalks, sprouts, seeds, etc.)37,38. In addition, the broccoli florets found in your grocery store have less glucoraphanin than broccoli sprouts and seeds. It has been determined that broccoli seeds have significantly higher levels of glucoraphanin than both broccoli florets and broccoli sprouts, which further translates into higher Phase II Enzyme Inducer Activity (Figure 6, data not published). The proprietary truebrocTM seeds are grown in California and the glucoraphanin is water-extracted under patented cGMP processes in Canada with complete traceability field to product. Almost twenty years of research into harvesting and extraction has resulted in truebrocTM, standardized to 13% glucoraphanin, the highest level on the market. A 230 mg daily serving of truebrocTM contains as much glucoraphanin as 10 ounces of regular broccoli – more than half a pound!

AS NATURE INTENDED IT Plants do not naturally produce sulforaphane; they make the components required to synthesize sulforaphane when the conditions are right (glucoraphanin and myrosinase). This is because sulforaphane is known to be highly unstable, especially in high temperature conditions and as such would degrade over time if the plant made and stored it.39,40 Therefore one of the main reasons to standardize to glucoraphanin versus converting it to sulforaphane is because of the stability of glucoraphanin, especially in high heat conditions, with only ~ 5% losses as a result of cooking or steaming41. Sulforaphane stability issues can be further exacerbated in finished products such as supplements and skin care products, where temperature sensitivity can translate into reduced shelf-life, especially in hotter environments. It was shown that the degradation rate of sulforaphane increased by a factor of nearly 3.1 for every 10 °C increase in temperature with complete degradation seen after 30 days in a conventional pharmaceutical cream formulation42. Advanced spray drying techniques may help stabilize sulforaphane for extended periods of time but will add cost while ultimately diluting the product43. In addition, the bitter flavor profile of sulforaphane is also not easy to mask, making glucoraphanin a more pleasant candidate for use in foods, beverages, and the like.

TRUEBROCTM BIOEQUIVALENCY STUDIES PROVE IT’S EFFECTIVE The patented harvest and extraction processes used for truebrocTM builds on the original work performed at Johns Hopkins University. The majority of the original JHU research utilized broccoli sprouts standardized to the levels of glucoraphanin compared to broccoli florets. Further analyses led to the discovery that broccoli seeds were even higher in glucoraphanin than sprouts, providing the foundation for the seed cultivation that would lead to the unique proprietary broccoli seeds extracted to make truebrocTM. A crossover study was performed at Johns Hopkins University to determine if the conversion efficiency of glucoraphanin to sulforaphane is the same when truebrocTM is used compared to the 3-day old broccoli sprout preparations that had been used in many JHU studies44. The urine samples of 20 adults were analyzed for sulforaphane metabolites 4 separate times using two different doses of two different glucoraphanin preparations; two from truebrocTM and two from 3-day old broccoli sprouts prepared in the same manner as used in all of the JHU studies. When the values for the 20 subjects were averaged, the percent conversions of glucoraphanin to sulforaphane for the two preparations (truebrocTM and broccoli sprouts) were statistically the same and in the range of 11% to 13% for the lower dose and 8% to 10% for the higher dose. This study demonstrated the bioavailability of glucoraphanin from truebrocTM is equivalent to that of the 3-day-old broccoli sprout preparations that have been used in all Johns Hopkins University studies, thus providing evidence that all of the results seen in the Hopkins studies would be similar when using truebrocTM.

CONCLUSION: TRUEBROCTM IS A PREMIER LONG-LASTING INDIRECT ANTIOXIDANT THAT SUPPORTS CELL INTEGRITY AND PROTECTS AGAINST FREE RADICAL DAMAGE truebrocTM has many advantages over more widely consumed and recognizable antioxidants because the glucoraphanin in truebrocTM is converted into sulforaphane by gut microflora and, once converted, it boosts the body’s own Phase II Enzyme System to help protect cells. This system helps to reduce oxidative stressors and enhance the removal of environmental toxins, increasing cellular antioxidant defense and contributing to healthy organ function.

The addition of truebrocTM benefits brand owners and manufacturers seeking a “true” antioxidant with beauty-from- within, sports nutrition, cellular protection and overall heightened antioxidant capabilities.

WHY GLANBIA NUTRITIONALS? Glanbia Nutritionals 2840 Loker Avenue East Glanbia Nutritionals is a global leader in ingredient solutions, Carlsbad, CA 92010 providing precision premixes, amino acids, vitamins, minerals, specialty ingredients and colors for the food, beverage and +1 800 735 8137 supplement industries. Built on our reputation for outstanding [email protected] quality and service, we deliver formulation and ingredient glanbianutritionals.com expertise that help our customers optimize their products and © 2015 Glanbia Nutritionals. All rights reserved. propel them to greater success.

REFERENCES

1. Mintel NPD 2015 2. Wojcik et al; Curr Med Chem. 2010;17(28):3262-88 3. Moneim; Curr Alzheimer Res. 2015;12(4):335-49 4. Phaniendra et al; Indian J Clin Biochem. 2015 Jan;30(1):11-26; 5. Kirkham & Rahman; Pharmacol Ther. 2006 Aug;111(2):476-94; 6. Jiang et al; Ann Allergy Asthma Immunol. 2014 Aug;113(2):137-42; 7. Phillips et al; Antioxid Redox Signal. 2010 Mar 15;12(6):743-85 8. Dandekar et al; Methods Mol Biol. 2015;1292:205-14 9. Sousa et al; Int J Food Sci Nutr. 2014 Mar;65(2):151-63; 10. Yavari et al; Asian J Sports Med. 2015 Mar;6(1):e24898 11. Sheweita & Sheikh; Curr Drug Metab. 2011 Jul;12(6):587-93; 12. Peng et al; Biomed Res Int. 2014;2014:831841 13. Aquilano et al; Front Pharmacol. 2014 Aug 26;5:196; 14. Saharan & Mandal; J Alzheimers Dis. 2014;40(3):519-29; 15. Huang et al; Semin Cell Dev Biol. 2012 Sep;23(7):738-44 16. Bocci & Valacchi; Front Chem. 2015 Feb 2;3:4; 17. Stefanson & Bakovic; Nutrients. 2014 Sep 19;6(9):3777-801 18. Gao et al; PNAS.2001; 98(26); 15221-15226 19. Padayatty et al; Ann Intern Med. 2004;140:533-537 20. Watanabe et al; Biomed Res Int. 2014;2014:140165; 21. Batinic-Haberle et al; Antioxid Redox Signal. 2014 May 20;20(15):2372-415 22. No et al; J Cancer Prev. 2014 Jun;19(2):111-7 23. Galal et al; Curr Top Med Chem. 2015;14(24):2802-21 24. Gao et al; PNAS.2001; 98(26); 15221-15226 25. van Muiswinkel & Kuiperij; Curr Drug Targets CNS Neurol Disord. 2005 Jun;4(3):267-81; 26. Shih et al; J Biol Chem. 2005 Jun 17;280(24):22925-36 27. Petri et al; Neurol Res Int. 2012;2012:878030; 28. Itoh et al; J Clin Biochem Nutr. 2015 Mar;56(2):91-7 29. Zhang et al; Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2399-403; 30. Fahey & Talalay; Food Chem Toxicol. 1999 Sep-Oct;37(9-10):973-9 31. Conzatti et al; Nutr Hosp. 2014 Nov 30;31(2):559-69 32. James et al; Nutr Rev. 2012 Nov;70(11):654-65 33. Gasper et al; J Nutr. 2007;137:1718–1724 34. Fowke et al; Carcinogenesis, 27(10), 2096–2102, 2006 35. Riedl et al; Clin Immunol. 2009;130:244–251 36. Brassica and Wakefield Research; Online omnibus survey, March 2015; not published 37. Saha et al; Mol Nutr Food Res. 2012 Dec;56(12):1906-16; 38. Atwell et al; Mol Nutr Food Res. 2015 Mar;59(3):424-33 39. Van Eylen et al; J Agric Food Chem. 2007 Mar 21;55(6):2163-70; 40. Liang & Yuan; Crit Rev Biotechnol. 2012 Sep;32(3):218-34 41. Yuan et al; J Zhejiang Univ Sci B. 2009 August; 10(8): 580–588 42. Franklin et al; Drug Dev Ind Pharm. 2014 Apr;40(4):494-502 43. Wu et al; Carbohydr Polym. 2014 Feb 15;102:497-503 44. Paul Talalay, P.I., Conversion of Broccoli Seed Glucosinolates to Isothiocyanates – Dietary Supplements, “submitted 08/28/2014