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In this paper we detail the development of Mannatech’s antioxidant, Ambrotose AO™, which culminated in a small human clinical trial. Consumption of two capsules of Ambrotose AO™ per day was found to increase a blood serum marker of oxidative protection by 37.4%, which is over twice the increase caused by consumption of an additional fi ve servings of fruits and vegetables each day (Figure 1).1

BackgroundThe body has an elaborate network of innate molecules and ingested compounds designed to maintain a balanced oxidative state, which is important for optimal function and health (Figure 2).2

Oxidative stress, a condition characterized by an imbalance between pro-oxidants and antioxidants within the body, has been linked to the aging process. A growing body of evidence suggests that a diet high in fruits and vegetables helps prevent oxidative stress. In fact, antioxidants—chemicals able to quench or neutralize free radicals—are thought to be responsible for many of the benefi ts that fruits and vegetables confer. Because few eat suffi cient amounts of fruits and vegetables, supplementation with antioxidants would seem to be the key to providing the benefi ts offered by diets high in fruits and

Innovations in the Development of a Superior Antioxidant: Ambrotose AO™O™OBill McAnalley, PhD; Tom Gardiner, PhD; Alyssa Summey; Shayne McAnalley; Eileen Vennum, RAC

Additional 5Servings ofFruits andVegetables

1 CapsuleAmbrotose AO™

2 CapsulesAmbrotose AO™

Figure 1.Comparsion of Percent Change in Human Serum ORACß-PE

Values Following Consumption of 5 Additional Fruits andVegetables* or Ambrotose AO™ Finished Product**

* Cao G, Booth SL, Sadowski JA, et al. AM JClin Nutr. 1998; 68(S):1081-1087.**Boyd S, Ford C, Koepke CM, et al. GlycoScience & Nutrition 2003.4(6):1-6.

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EXTERNAL EDITORIAL BOARD

Tom Gardiner, PhDGlobal Health Safety Environment and Regulatory Affairs CoordinatorShell Chemical Company (Retired) Houston, Texas

Robert K. Murray, MD, PhDProfessor (Emeritus)Department of BiochemistryUniversity of TorontoToronto, Ontario, Canada

Alice Johnson-Zeiger, PhDProfessor of Biochemistry (Retired) University of Texas Health Center Tyler, Texas

James C. Garriott, PhD, D-ABFTProfessor (Clinical Adjunct Faculty)University of Texas Health Science CenterConsulting ToxicologistSan Antonio, Texas

MANNATECH INCORPORATED INTERNAL CONTRIBUTING AND CONSULTING EDITORSStephen Boyd, MD, PhD, FRSMBill McAnalley, PhD

TECHNICAL STAFFGary Carter Barbara KinseyMary Wood

GRAPHIC ARTISTSBruce PeschelJohn Pavel

SCIENCE EDITORC. Michael Koepke, RAC

MANAGING EDITORJane Ramberg, MS

EDITOR IN CHIEFEileen Vennum, RAC

The Offi cial Scientifi c Publicationof the Research & DevelopmentDepartment of Mannatech Inc.Coppell, Texas, USA. © 2004. All rights reserved.

www.mannatech.com

Glossary Terms on Page 6References on Page 7

Volume 1, January 1, 2004Volume 1, January 1, 2004

Page 2—Mannatech R&D Report, Vol. 1. January 1, 2004

vegetables. However, caution should be observed when choosing an antioxidant supplement.

Figure 2.

Problems with Antioxidant SupplementationSupplementation with high doses of single antioxidant supplements or combinations of a few antioxidants has proven ineffective in some cases and harmful in others. The doses used in these failed trials differed greatly from amounts found in fruits and vegetables and often included only either lipid- or water-soluble antioxidants.

There are other key differences between foods with abundant antioxidant nutrients and antioxidant supplements. Antioxidants in the body do not function independently. Whole foods provide numerous compounds that can enhance antioxidant function, while supplements often provide pure forms of isolated nutrients that lack the accompanying nutrients necessary for proper function. Also,

nutrients in foods can be released more slowly than those in supplement form. The large doses of highly bioavailable antioxidants may result in a brief spike in blood plasma antioxidant levels (which may exceed healthy levels) followed by a return to defi cient levels (Figure 3).

Figure 3.

In contrast, foods may offer more subtle and sustained protection. The high doses of isolated nutrients that are often involved in supplementation can have undesirable consequences. Because free (unbound) vitamin A damages cells, symptoms of toxicity arise when the amount of vitamin A ingested overwhelms the number of proteins available to bind and transport it throughout the body. Toxic levels of vitamin A are unlikely to develop in individuals who derive their vitamin A from a balanced diet. Supplementation, on the other hand, makes an overdose of vitamin A quite possible. Similarly, supplemental forms of beta-carotene can be dangerous, but eating too much beta-carotene from food sources, though it may cause the skin to appear yellow, is harmless.3 Researchers found that whole tomato powder but not lycopene, a carotenoid found in tomatoes, inhibited prostate carcinogenesis in rats, demonstrating the functionality of a whole-food supplement and the lack of effi cacy of a high dose of an isolated nutrient.4

Since foods have been effective in combating oxidative stress, and supplements generally have not, reconciling the differences between the two and minimizing their shared vulnerability in processing and handling became our focus during the formulation of Ambrotose AO™. Our considerations are summarized in Table 1.

Importance of BalanceNumerous studies have demonstrated that cooperation among antioxidant nutrients is essential for proper function.5,6 Without such cooperation, free radicals can become concentrated in certain areas within the body, creating a capacitor effect, meaning that antioxidants fuel localized chain reactions, harming rather than protecting

Comparison of Foods Rich in Antioxidants andAntioxidant Supplements

Foods Rich in Antioxidants

Have been shown to protect from oxidative stress.

There may be no single active compound. In fact, studies point to the effec-tiveness of combinations rather than single nutrients. Foods can provide these effective combinations in low but balanced, highly effective amounts.

Can offer a slower, sustained delivery of nutrients.

Symptoms of toxicity are unlikely to develop in individuals who derive nutrients from a balanced diet.

Can suffer tremendous nutrient losses during cultivation, cooking, processing and storage.

Antioxidant Supplements

Have often failed to show any protective effect against oxidative stress and have been demonstrated to be dangerous in some cases.

Can provide high doses of isolated nutrients (a “magic bullet” approach), with none of the accompanying compounds that enable function.

Generally lack a controlled release.

High doses of readily available nutrients in supplement form can overwhelm the body and upset the natural balance.

Can suffer tremendous nutrient losses during processing, handling and storage (e.g., exposure to heat or oxygen).

Effect in Humans

Dosage of Active Compound

Delivery of Nutrients

Potential for Toxicity

Vulnerability in Processing

minimum effectiveconcentration (MEC)for adverse response

minimum effectiveconcentration (MEC)for desired response

Therapeutic Window for Antioxidants

therapeuticwindowonset of

effect

durationof actionLe

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Blo

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Pla

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Time

Table 1.

Scheme for Balanced Cellular Oxidative Status

Yu. BP. In: Cutler RG. Packer L. Bertram J. and Mori A. Editors. Oxidative Stress and Aging.Berkgauser Verlag, Switzerland. 1995:331-342.

Oxidative Reactions

Reactive oxygen species generation

Oxidation of proteins, lipids, nucleic acids, and sugars

Transition metal catalysis

Antioxidants

Regulation of metals

Scavenger enzymes

Damage repair

Elimination of oxidatively damaged by-products

Defense Systems

Mannatech R&D Report Vol. 1, January 1, 2004—Page 3

cells.7,8 Hence, the idea of balance extends from a balance between pro-oxidants and antioxidants within the body to a balance among antioxidants themselves (Figure 4).

Figure 4.

When formulating antioxidant supplements, achieving balance has been impeded because scientists have lacked the tools to analyze interactions among antioxidant compounds with varying solubilities. Current methods for assessing the antioxidant potential of samples, the best of which are fl uorescence-based ORAC (oxygen radical absorbance capacity) methods, do not allow lipid- and water-soluble samples to be tested at the same time. The ORAC

fl method is appropriate for assaying the antioxidant

capacity of water-soluble samples.9 The ORACfl -lipo

method was designed to assay lipid-soluble samples10 but often does not take into account water-soluble elements. These currently used methods also involve an initial extraction that reduces or excludes less soluble (but perhaps functionally important) parts of a sample. At best, lipid- and water-soluble portions of samples are separated and analyzed individually, but this separation prevents analysis of meaningful interactions. Since antioxidants in the body often work in concert and include both lipid- and water-soluble compounds, an assay that allows both types of antioxidants to interact (potentially cooperating or interfering with each other) would provide a more accurate model of antioxidant activity in humans.

The ORACo method, which uses an oxygen-specifi c probe,

was created to address problems with current industry standards. Because of its unique solvent system and sample preparation technique, the ORAC

o method, which

simultaneously measures the activities of lipid- and water-soluble compounds, allows the detection of interactions that cannot be measured through the use of fl uorescence-based antioxidant assays.

Formulation of Mannatech’s Ambrotose AO™

Balance, with the aim of optimal performance in humans, was the controlling idea behind the formulation of the MTech AO Blend™. Synergy (meaning the activity of a mixture as a whole is greater than that of the sum of its parts) was discovered between quercetin and mixed tocopherols using the ORAC

o method, demonstrating the type of cooperation

necessary for effective radical quenching in humans. Roughly two times the expected net antioxidant activity was found at the optimal ratio of the two compounds.

Mixed tocopherols are lipid-soluble, and quercetin is only slightly soluble in water. Focus was then shifted to the inclusion of water-soluble antioxidants. Based on their known benefi ts, green tea extract and grape extract were selected, as well as freeze-dried Australian bush plum, which has the highest amount of vitamin C per gram of any known natural source,11 and Phyt•Aloe® complex, which provides dehydrated fruits and vegetables. Numerous samples of quercetin, mixed tocopherols, grape extract and green tea extract were individually assayed for activity using the ORAC

fl method. The sample with the highest

activity for each component was included in blends, and the ratio of components was optimized using the ORAC

o

method, the assay best suited to analyze interactions among various compounds.

Oxygen Radical Absorption Capacity of the MTech AO Blend™

The resulting in-process MTech AO Blend™, the active component of Ambrotose AO™, was run using the ORAC

o,

ORACfl and ORAC

fl -lipo assays. Results were reported in μmol

Trolox Equivalents (TE)/g or /mL. The ORACfl assay detects

only the activity of water-soluble components. It measured only the activity of green tea extract, grape extract, bush plum, Phyt•Aloe®, and part of quercetin’s activity, but excluded the contribution of mixed tocopherols entirely. The ORAC

fl -lipo detects only the activity of lipid-soluble

components. It measured mixed tocopherols and part of quercetin’s activity, but excluded the contributions of the water-soluble components listed above. In comparison, the ORAC

o picks up the activity of both water- and lipid-soluble

components and was able to detect the synergistic activity of all components of the MTech AO Blend™. The MTech AO Blend™ measured a whopping 17,454 ORAC

o units!

Mother Nature Knows BestPrevious failures with high-dose antioxidant supplemen-tation have shown that more of pure substances is not necessarily better. For example, large doses of purifi ed, individual vitamins can lead to very high, followed by very low, levels of those vitamins in the blood and tissues that need them (Figure 3). As a result, antioxidant nutrients are often not available when they are needed for necessary biological functions, or they can be present in toxic amounts. A steady, sustained bioavailability of a suffi cient

Page 4—Mannatech R&D Report, Vol. 1. January 1, 2004

amount of antioxidants is critical for providing protection against oxidative tissue damage. In other words, antioxidant supplements should closely resemble the antioxidant-rich foods that gradually release suffi cient vitamins and minerals during digestion and absorption (Figure 5).

Figure 5.

In order to create an antioxidant supplement that more closely resembles food, Ambrotose® complex was included in the antioxidant formula at a ratio of Ambrotose® complex:MTech AO Blend™ of 2:1. This mixture was chosen, in part, because many of the gums contained in Ambrotose®

complex have been used to achieve the sustained release of ingredients in other compounded mixtures.12,13 When antioxidants are taken with glyconutritional sugars, there is a gradual release that allows antioxidants to act as radical scavengers throughout the digestive tract.14 Inclusion of Ambrotose® complex in the formula also provides glyconutrients to support cell-to-cell communication, as well as enhanced antioxidant functionality.

Processing and handling techniques can dramatically change the presence and activity of a food’s constituents. To illustrate, green, black and oolong teas all come from the same evergreen tree, Camellia sinensis. The difference in the properties and fl avors of the teas results from the processing technique employed. Compared to black tea, green tea contains up to ten times the levels of polyphenols, which are thought to be responsible for many health-promoting effects. Thus, special attention was paid to the manufacturing process of Ambrotose AO™ in order to preserve the activity of the MTech AO Blend™. Once again, nature served as a guide.

In nature, nutrients within plant sap are not exposed as readily to oxygen, and so they are protected from oxidation (Figure 6).

Similarly, Ambrotose AO™ was manufactured in a way which ensures that the antioxidant ingredients retain their potency

Figure 6.

throughout the manufacturing process and thereafter in storage. A roller compaction manufacturing process was designed to help limit the exposure of ingredients to oxygen (Figure 7).

The compaction process also renders the fi nal product ingredients more homogeneous in particle size and distribution within the mixture,15 and is designed to enhance the ability of Ambrotose® complex to provide a more sustained release of antioxidant ingredients.

Figure 7.

The Need to Demonstrate Clinical EffectivenessIt should be noted that in vitro assays are merely tools. High radical-quenching ability in a test tube does not necessarily translate into effective radical quenching in the body. Even the most sophisticated in vitro antioxidant assay can only be used to predict the action of an antioxidant formula on the body. Also, radical-quenching ability, while important, does not completely refl ect antioxidant ability (Figure 2). The only way to know an antioxidant’s true effectiveness is to examine its action in people.

MEC fordesiredresponse

MEC foradverseresponse

Benefit of a Controlled Release

therapeuticwindowonset of

effect

durationof action

Leve

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Mannatech R&D Report Vol. 1, January 1, 2004—Page 5

Measurement of an Individual’s Oxidative Stress StateAchieving a meaningful measurement of oxidative stress poses a challenge. Even the best single measure of antioxidant status cannot give a clear picture of what is happening throughout the body. Living organisms have an elaborate network of defense against oxidative stress. A wide array of innate molecules and ingested compounds work together to prevent the formation of radicals, quench radicals when they form, and repair the damage that radicals cause to cells. Researchers have concluded that a battery of tests is necessary to assess an individual’s oxidative stress state adequately.16

Tests measuring levels of oxidative damage and protection are the best candidates for inclusion in a battery of tests. The most widely used tests that assess oxidative damage measure products of damage to cells in bodily fl uids (i.e., blood, urine or breath). Several methods have been developed to measure the protection offered by blood serum, and a comparison of these methods highlighted the ORAC method as having considerable advantages over the others.17

Researchers sometimes examine levels of antioxidants and other compounds within the body. Examinations of vitamin and mineral levels in the blood or even the skin can confi rm the presence of compounds but provide no evidence of their activity within the body. When assessing an individual’s oxidative stress state, an examination of activity rather than presence yields more valuable information since all antioxidants can behave as pro-oxidants under certain conditions. Some of the more dramatic examples of pro-oxidant activity of an antioxidant in humans have been shown with beta-carotene.

Clinical Effectiveness of Ambrotose AO™

In a pilot human clinical trial conducted by Mannatech’s Research and Development Department, the effects of escalating doses of Ambrotose AO™ were evaluated based ™ were evaluated based ™

upon a number of parameters in which samples from fasting subjects were collected and analyzed by independent contract laboratories, and statistics were performed by an independent contract fi rm.16 A battery of tests was selected in order to assess levels of both oxidative damage and protection.

An increase in fasting serum ORACß-PE values, which

indicates increased oxidative protection, was found at all three doses: 19.1% at 500 mg per day, 37.4% at 1.0 g per day, and 14.3% at 1.5 g per day. In a published study of healthy adults examining the effects of increasing fruit and vegetable consumption from the usual fi ve to an experimental ten servings per day over two weeks, an increase in fasting serum ORACß-PE

values of roughly 13% was reported.1 Pilot

test data indicates that on average over twice this increase, 37.4%, was found with 1.0 g (two capsules) per day of Ambrotose AO™ over the same time period (Figure 1).

The increases observed in fasting serum ORACß-PE values

among those taking Ambrotose AO™ are strong indicators ™ are strong indicators ™

of a sustained effect. The increased protection observed in those taking Ambrotose AO™ was measured at least 12 ™ was measured at least 12 ™

hours following supplementation. In a study of the effects of beverages with high ORAC values (including spinach, red wine, ascorbic acid or strawberries), increases in serum antioxidant capacity were brief in duration, often returning toward or below baseline levels within four hours following beverage consumption.18 Thus, some fruits and vegetables in beverage form may not offer such sustained antioxidant protection as was observed following supplementation with

Ambrotose AO™.

Escalating doses of Ambrotose AO™ also caused a trend of decreased markers of lipid oxidative damage (urinary lipid hydroperoxides/creatinine): 12.2% at 500 mg per day, 15.0% at 1.0 g per day, and 17.0% at 1.5 g per day. As ozone levels rise, levels of markers of oxidative damage would be expected to rise as well. Thus, the lack of signifi cant changes in markers of lipid and DNA damage

(urinary alkenal and 8-OHdG levels, respectively) despite decreasing air quality throughout the study also suggests protection. Decreasing air quality may also explain the smaller increase in protection observed in subjects taking three capsules compared to two capsules of Ambrotose AO™

per day.

DosageThe optimal dosage of Ambrotose AO™ based upon serum ORACß-PE

values in healthy adults appears to be 1.0 g (two capsules). However, stressed individuals may benefi t from higher doses of antioxidants.19

Because of the lack of established upper limits for the other ingredients in Ambrotose AO™, vitamin E was examined in order to illustrate safe levels of intake. A safe and optimal daily intake of vitamin E for men and women ranges between 400 and 1200 IU.20 Hence, one could take up to 66 capsules of Ambrotose AO™, which provides 18 IU of vitamin E per capsule, and still remain within the optimal levels of vitamin E intake. Those taking Mannatech’s vitamin and mineral support, Glycentials®, which provides 400 IU of vitamin E per day, could take up to 44 capsules of Ambrotose AO™ and remain within the optimal range of intake for vitamin E. Those with high blood pressure and those taking anticoagulants should not consume more than 400 IU of vitamin E daily without consulting their physician.20

When antioxidants are taken

with glyconutritional sugars,

there is a gradual release that

allows antioxidants to act as

radical scavengers throughout

the digestive tract.

Page 6—Mannatech R&D Report, Vol. 1. January 1, 2004

SummaryControl of oxidative stress is crucial to preserving health and longevity.21 Foods high in antioxidant nutrients have been shown to protect against oxidative stress. Antioxidant supplementation with high doses of antioxidants has often proven ineffective and sometimes even dangerous. With this in mind, we created an antioxidant supplement that more closely resembles food, Ambrotose AO™, which was designed to contain a balance of antioxidants, to release them slowly to sustain protection, and to protect the active compounds by limiting their exposure to oxygen. When its antioxidant effect was examined in humans, 1.0 g (two capsules) of Ambrotose AO™ provided on average over twice the protection offered by an additional fi ve servings of fruit and vegetables to the diet based upon fasting serum ORACß-PE

values.

Glossary Terms

antioxidantAn agent that inhibits oxidation or reactions promoted by oxygen or peroxides; its actions are often described as radical quenching or neutralizing. It should be noted that under certain conditions, antioxidants can function as pro-oxidants, compounds that promote oxidation.

assayTest used to determine the amount of a particular constituent of a mixture or the activity of a compound.

bioavailabilityThe degree of physiological availability (proportion of the administered dose that is absorbed into the bloodstream or available for use within the body) of a compound.

carotenoidAny of a group of red, yellow or orange pigments that are found in foods such as carrots, sweet potatoes and leafy green vegetables, as well as in some animal tissues.

free radicalA compound with one or more unpaired electrons. Free radicals are unstable and react readily with other molecules. They may be short-lived, highly active intermediates involved in various reactions in living tissue.

homogeneousSimilar or the same in structure or composition.

hydroxyl radicalA highly oxidizing type of free radical that attacks all biomolecules.

innateDerived from factors possessed at birth; inherent; inborn.

in vitroStudies done “in glass” (in the test tube). These tests are used to predict the effects a compound will have in “whole” animals or humans.

lipidA group of biomolecules that are somewhat soluble in water and very soluble in many organic solvents. Types of lipids include neutral fats (triglycerides), phospholipids, glycolipids, sterols and steroids, waxes and various terpenes.

serumFluid, non-cellular component of blood.

synergyA cooperative interaction wherein the effect of the combination is greater than the sum of the effects of the components.

Mannatech R&D Report Vol. 1, January 1, 2004—Page 7

References 1. Cao G, Booth SL, Sadowski JA, et al. Increases in human plasma antioxidant capacity after consumption of controlled

diets high in fruit and vegetables. Am J Clin Nutr. 1998;68(5):1081-1087.

2. Yu BP. Modulation of Oxidative Stress as a means of life-prolonging action of dietary restriction. In: Cutler RG, Packer L, Bertram J, and Mori A, editors. Oxidative Stress and Aging. Birkhauser Verlag,Basel, Switzerland.1995:331-342.

3. Whitney E, Rolfes S. Understanding Nutrition. 9th Edition. Belmont, Ca.: Wadsworth/Thomson Learning, 2002.

4. Boileau TW, Liao Z, Kim S, et al. Prostate carcinogenesis in N-methyl-N-nitrosourea (NMU)-testosterone-treated rats fed tomato powder, lycopene, or energy-restricted diets. J Natl Cancer Inst. 2003;95(21):1578-1586.

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6. Kontush A, Finckh B, Karten B, et al. Antioxidant and prooxidant activity of alpha-tocopherol in human plasma and low density lipoprotein. J Lipid Res. 1996;37(7):1436-1448.

7. Bowry VW, Stocker R. Tocopherol-mediated peroxidation. The pro-oxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J Am Chem Soc. 1993;115(14):6029-6044.

8. Thomas SR, Stocker R. Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis. Free Radic Biol Med. 2000;28(12):1795-1805.

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10. Huang D, Ou B, Hampsch-Woodill M, et al. Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer. J Agric Food Chem.2002;50(7):1815-1821.

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13. Billa N, Yuen KH, Khader MA, et al. Gamma-scintigraphic study of the gastrointestinal transit and in vivo dissolution of a controlled release diclofenac sodium formulation in xanthan gum matrices. Int J Pharm. 2000;201(1):109-120.

14. Spiller GA, editor. CRC Handbook of Dietary Fiber in Human Nutrition. 3rd Edition. New York, New York: CRC Press, 2001.

15. Wells J, Rubinstein M, ed. Pharmaceutical Technology Tableting Technology Volume 2 (Compression). Chicester: Ellis Pharmaceutical Technology Tableting Technology Volume 2 (Compression). Chicester: Ellis Pharmaceutical Technology Tableting TechnologyHorwood, 1993.

16. Pryor WA, Boyd T, Boyd D, ed. Bioassays for Oxidative Stress Status (BOSS). 1st Edition. New York, NY: Elsevier, 2001.

17. Cao G, Prior RL. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin Chem. 1998;44(6 Pt 1):1309-1315.

18. Cao G, Russell RM, Lischner N, et al. Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. J Nutr. 1998;128(12):2383-2390.

19. Schmidt MC, Askew EW, Roberts DE, et al. Oxidative stress in humans training in a cold, moderate altitude environment and their response to a phytochemical antioxidant supplement. Wilderness Environ Med. 2002;13(2):94-105.

20. Lieberman S, Bruning N. The Real Vitamin and Mineral Book. 2nd Edition. Honesdale, Pa.: Paragon Press, 1997.

21. Ochi H, Cheng RZ, Kantha SS, et al. The JaICA-genox oxidative stress profi le—an overview on the profi ling technique in the oxidative stress assessment and management. Biofactors. 2000;13(1-4):195-203.

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