US20180015070A1

US20180015070A1 - Methods and compositions for upregulating endogenous antioxidant systems

Info

Publication number: US20180015070A1
Application number: US15/643,329
Authority: US
Inventors: Mark Brown; John Cuomo; Jeremy Tian
Original assignee: Usana Health Sciences Inc
Current assignee: Usana Health Sciences Inc
Priority date: 2016-07-06
Filing date: 2017-07-06
Publication date: 2018-01-18
Also published as: US20180008573A1; US20180007945A1; US10632101B2

Abstract

A nutritional supplement for reducing free radical damage is disclosed. The nutritional supplement includes a first vehicle comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture and a second vehicle comprising a mineral mixture. The upregulating compound mixture is configured to upregulate an endogenous antioxidant system to protect against free radical damage. The upregulating compound mixture includes alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The endogenous antioxidant system includes transcription factors such as Nrf2, NF-κB, PPARα, PPARβ/δ, and PPARγ that promote transcription of antioxidant genes.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/359,106 (Attorney Docket No. 11224.33), filed Jul. 6, 2016, entitled “METHODS AND COMPOSITIONS FOR UPREGULATING ENDOGENEOUS ANTIOXIDANT SYSTEMS,” and claims priority to U.S. Provisional Patent Application No. 62/359,113 (Attorney Docket No. 11224.34), filed Jul. 6, 2016, entitled “METHODS AND COMPOSITIONS FOR REDUCING DAMAGE ASSOCIATED WITH OXIDATIVE PHOSPHORYLATION,” and claims the benefit of U.S. Provisional Patent Application No. 62/359,120 (Attorney Docket No. 11224.35), filed Jul. 6, 2016, and entitled “METHODS AND COMPOSITIONS FOR SUPPORTING ENDOGENOUS SYSTEMS RELATED TO LIFE SPAN,” the entire disclosures of which are hereby incorporated by reference.

BACKGROUND

This disclosure pertains to methods and compositions for upregulating endogenous antioxidant systems. More particularly, it pertains to nutritional supplements configured to upregulate endogenous antioxidant systems. Additionally, it pertains to methods of manufacturing these nutritional supplements and methods of administering these nutritional supplements. The nutritional supplements can comprise various active ingredients including antioxidant compounds and compounds, which upregulate endogenous antioxidant systems (e.g., compounds such as alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin).
Conventional nutritional supplements often comprise nutrients such as vitamins, minerals, dietary elements, fatty acids, and other vital nutrients. These nutrients often include compounds such as vitamins that are vital for growth and development, but cannot be produced by the body. Sometimes nutritional supplements can include exogenous antioxidants such as vitamin C, vitamin E, beta-carotene, and other carotenoids that provide the body protection against free radicals, provided that the exogenous antioxidants are absorbed and retained by the body in sufficient concentrations. Because the body cannot produce some of these exogenous antioxidants and because they can be excreted by certain systems in the body, these exogenous antioxidants must be regularly consumed to provide ongoing protection against free radicals. In addition to systems to utilize exogenous antioxidants, the body also comprises endogenous antioxidant systems that can help defend against free radical damage. These exogenous antioxidant systems include endogenous antioxidants such as glutathione and antioxidant enzymes such as glutathione reductase, glutathione peroxidases, glutathione-S-transferases (GST), superoxide dismutase (SOD), NAD(P)H Dehydrogenase, Quinone 1 (NQO-1), Heme Oxygenase 1 (HO-1), and Glutamate-Cysteine Ligase, Catalytic Subunit (GCL).
Although conventional nutritional supplements provide a variety of benefits, conventional nutritional supplements are not necessarily without their shortcomings. For example, while conventional nutritional supplements may provide exogenous antioxidants, conventional nutritional supplements do not spur the body to upregulate its own endogenous antioxidant systems. Also, while conventional nutritional supplements may provide exogenous antioxidants, the conventional nutritional supplements are not configured to provide the long-lasting benefit of an increase in endogenous antioxidants.
Thus, while some conventional nutritional supplements currently exist, challenges still exist, including those listed above. Accordingly, it would be an improvement in the art to improve or replace current techniques and/or formulations.

BRIEF SUMMARY

The present application discloses compositions and methods for upregulating endogenous antioxidant systems. In some embodiments, compositions may include a nutritional supplement for reducing free radical damage that comprises an upregulating compound mixture configured to upregulate an endogenous antioxidant system, an exogenous antioxidant mixture; and a mineral mixture. The upregulating compound mixture may include one or more of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The exogenous antioxidant mixture may comprise one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.
In some embodiments, the nutritional supplement may comprise a first vehicle. In some embodiments the first vehicle may comprise an upregulating compound mixture configured to upregulate an endogenous antioxidant system, and an exogenous antioxidant mixture. In some embodiments, the nutritional supplement may comprise a second vehicle. In some embodiments, the second vehicle may comprise a mineral mixture. The first and second vehicle may comprise a single solid tablet. The upregulating compound mixture may include one or more of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The exogenous antioxidant mixture can comprise one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.
In some embodiments, the methods for reducing free radical damage comprise administering a first vehicle, comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture, and administering a second vehicle comprising a mineral mixture, where the upregulating compound mixture is configured to upregulate an endogenous antioxidant system to reduce free radical damage. The endogenous antioxidant system can comprise a transcription factor such as Nrf2, NF-κB, PPARα, PPARβ/δ, and PPARγ. The transcription factor can promotes transcription of an antioxidant gene such as a Phase II gene, a NQO1 gene, a GCL gene, a sulfiredoxin 1 (SRXN1) gene, a thioredoxin reductase 1 (TXNRD1) gene, a HO-1 gene, a GST family gene, and an UDP-glucuronosyltransferase (UGT) family gene.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and other advantages and features of the disclosure can be obtained, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the disclosure and are not therefore to be considered to be limiting of its scope, the disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

FIG. 1 illustrates a model of upregulation of endogenous antioxidant systems;

FIG. 2A illustrates a chemical structure of alpha lipoic acid;

FIG. 2B illustrates a chemical structure of resveratrol;

FIG. 2C illustrates a chemical structure of curcumin;

FIG. 3A illustrates a chemical structure for epigallocatechin gallate (EGCG);

FIG. 3B illustrates a chemical structure of rutin;

FIG. 3C illustrates a chemical structure of quercetin;

FIG. 4 illustrates a chemical structure of hesperetin;

FIG. 5 illustrates fold-activation of PPARα for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 6 illustrates fold-activation of PPARα for Olivol®, rutin, quercetin, and hesperetin;

FIG. 7 illustrates fold-activation of PPARα for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin;

FIG. 8 illustrates fold-activation for a known PPARα agonist, GW590735;

FIG. 9 illustrates fold-activation of PPARδ for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 10 illustrates fold-activation of PPARδ for Olivol®, rutin, quercetin, and hesperetin;

FIG. 11 illustrates fold-activation of PPARδ for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin;

FIG. 12 illustrates fold-activation for a known PPARδ agonist, GW0742;

FIG. 13 illustrates fold-activation of PPARγ for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 14 illustrates fold-activation of PPARγ for Olivol®, rutin, quercetin, and hesperetin;

FIG. 15 illustrates fold-activation of PPARγ for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin;

FIG. 16 illustrates fold-activation for a known PPARγ agonist, rosiglitazone;

FIG. 17 illustrates fold-activation of Nrf2 for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 18 illustrates fold-activation of Nrf2 for Olivol®, rutin, quercetin, and Hesperetin.

FIG. 19 illustrates fold-activation of Nrf2 for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin;

FIG. 20 illustrates fold-activation for a known Nrf2 agonist, L-sulphoraphane;

FIG. 21 illustrates percent inhibition of human NF-κB in antagonist mode form for alpha lipoic acid, resveratrol, curcumin, and EGCG;

FIG. 22 illustrates percent inhibition of human NF-κB in antagonist mode form for Olivol®, rutin, quercetin, and hesperetin;

FIG. 23 illustrates percent inhibition of human NF-κB in antagonist mode form for a mixture of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin;

FIG. 24 illustrates an ideal Kaplan-Meier survival curve for a control population and a population exposed to an ideal test compound;

FIG. 25 illustrates a Kaplan-Meier survival curve for the worm population tested with N356 at 0.1 mg/ml concentration;

FIG. 26 illustrates a Kaplan-Meier survival curve for the worm population tested with N356 at 1.0 mg/ml concentration;

FIG. 27 illustrates a Kaplan-Meier survival curve for the worm population tested with N356 at 10 mg/ml concentration;

FIG. 28 illustrates a dose-dependent extension of lifespan for N356 at 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measured as a function of age at 20% mortality;

FIG. 29 illustrates a Kaplan-Meier survival curve for the worm population tested with N357 at the 0.1 mg/ml concentration;

FIG. 30 illustrates a Kaplan-Meier survival curve for the worm population tested with N357 at the 1.0 mg/ml concentration;

FIG. 31 illustrates a Kaplan-Meier survival curve for the worm population tested with N357 at the 10 mg/ml concentration;

FIG. 32 illustrates a dose-dependent extension of lifespan for N357 at 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measured as a function of age at 20% mortality;

FIG. 33 illustrates a Kaplan-Meier survival curve for the worm population tested with N108 (resveratrol) at the 0.1 mg/ml and 10 mg/ml concentrations;

FIG. 34 illustrates a Kaplan-Meier survival curve for the worm population tested with N198 (alpha lipoic acid) at the 0.1 mg/ml and 10 mg/ml concentrations;

FIG. 35 illustrates a Kaplan-Meier survival curve for the worm population tested with N347 (hesperidin) at the 0.1 mg/ml and 10 mg/ml concentrations;

FIG. 36 illustrates a Kaplan-Meier survival curve for the worm population tested with N104 (quercetin) at the 0.1 mg/ml and 10 mg/ml concentrations;

FIG. 37 illustrates a Kaplan-Meier survival curve for the worm population tested with N346 (rutin hydrate) at the 0.1 mg/ml and 10 mg/ml concentrations;

FIG. 38 illustrates fold-activation of Nrf2 by test solutions compared to control;

FIG. 39 illustrates fold-activation of Nrf2 by alpha lipoic acid compared to control;

FIG. 40 illustrates fold-activation of Nrf2 by quercetin compared to control; and

FIG. 41 illustrates fold-activation of Nrf2 by resveratrol compared to control.

DETAILED DESCRIPTION

Described herein are nutritional supplement compositions configured to upregulate endogenous antioxidant systems. In some embodiments, the methods and compositions disclosed in the present application include methods of preparing and compositions of nutritional supplements that comprise one or more of an upregulating compound mixture, an exogenous antioxidant mixture, and a mineral mixture. In other embodiments, the methods of preparing nutritional supplements and compositions of nutritional supplements comprise preparing nutritional supplements that comprise an upregulating compound mixture and an exogenous antioxidant mixture in a first part and a mineral mixture in a second part.
In some cases, damage by free radicals in cells of the body is linked to ageing and/or other acute and/or chronic diseases. Free radicals can include highly reactive atoms or molecules containing unpaired electrons. Free radicals can cause damage in biological systems when the free radical captures an electron from another molecule to pair with its unpaired electron. The molecule from which the electron was captured then becomes a free radical itself and seeks to capture another electron from another molecule, causing a chain reaction of free radical production.
Often, when a biological molecule loses an electron, it becomes damaged and ceases to function properly which can lead to damage within the cell. Free radicals can also cause cross-linking of biological structures such as cross-linking of DNA. DNA cross-linking may be damaging to the cell and may lead to ageing and diseases such as cancer. Free radical induced cross-linking may also be related to the formation of wrinkles, the formation of plaque in arteries leading to heart disease and stroke, and other chronic diseases.
In some cases, mitochondria are thought to be a main target of damage by free radicals. The production of energy through oxidative phosphorylation in the mitochondria provides the energy that the body needs to live, but also forms free radicals that may cause damage. While most of the free radicals generated during oxidative phosphorylation are neutralized, it is possible in some cases that the generated free radicals are not neutralized and can cause damage to the mitochondrial DNA and mitochondrial proteins. This damage to the mitochondrial DNA and mitochondrial proteins can lead to decreased mitochondrial efficiency. In some cases, the free radicals generated in the mitochondria can also leak into the cell and cause oxidative damage and/or death of the cell.
Some molecules, such as antioxidants, can inhibit the oxidation of other molecules and thereby neutralize the oxidizing effects of free radicals. Exogenous antioxidants such as thiols or ascorbic acid (vitamin C) can be found in certain foods and can work to counteract the effects of free radicals. In some cases, diets containing foods high in these exogenous antioxidants have been shown to improve overall health. Additionally, some nutritional supplements can include exogenous antioxidants such as vitamin C, vitamin E, beta-carotene, and other carotenoids that may provide the body some protection against free radicals. In some cases, the exogenous antioxidants must be absorbed and retained by the body in sufficient concentration to provide ongoing protection against free radicals. In other cases, some of these exogenous antioxidants must be regularly consumed because the body cannot produce them and because certain systems in the body work to metabolize or excreted them.
Various endogenous antioxidant systems in the body help the body to defend against free radical damage. In some embodiments, these endogenous antioxidant systems generate endogenous antioxidants such as glutathione that are configured to neutralize free radicals. In other embodiments, the endogenous antioxidant systems comprise endogenous antioxidant genes and/or enzymes that work to replenish or recharge the supply of endogenous antioxidants. In yet other embodiments, the endogenous antioxidant systems comprise endogenous antioxidant enzymes that themselves neutralize free radicals and/or reduce the damage caused by free radicals. These endogenous antioxidant genes and/or enzymes can include, but are not limited to, glutathione reductase, glutathione peroxidases, glutathione-S-transferases (GST), superoxide dismutase (SOD), NAD(P)H Dehydrogenase, Quinone 1 (NQO-1), Heme Oxygenase 1 (HO-1), and Glutamate-Cysteine Ligase, Catalytic Subunit (GCL), and proteins encoded by Phase II genes.
In some instances, these endogenous antioxidant systems can be upregulated to provide greater protection against free radical damage. Referring now to FIG. 1, a model of upregulation of an endogenous antioxidant system is illustrated. While various endogenous antioxidant systems may be upregulated, FIG. 1 illustrates a possible model for upregulation of endogenous antioxidant Phase II genes through a transcription factor, nuclear factor erythroid 2-related factor (Nrf2). The model can include a living cell 10 that comprises an outer membrane 12. The cell 10 can also comprise an inner nucleus 14 that is bounded by a nuclear membrane 16. The model can also include an inducer 20 that can signal upregulation of an endogenous antioxidant system (e.g., Phase II genes). The inducer 20 can include any suitable molecule such as a signaling molecule or an upregulating compound that can upregulate an endogenous antioxidant system. In some cases, the inducer 20 can cross the outer membrane 12 to signal upregulation of an endogenous antioxidant system. In other cases, the inducer 20 can interact with a receptor at the outer membrane 12 to signal upregulation of an endogenous antioxidant system. In yet other cases, the inducer 20 can interact with one or more signaling molecules and/or signaling complexes to signal upregulation of an endogenous antioxidant system.
In some embodiments, after the inducer 20 crosses the outer membrane 12, it acts to disrupt a complex formed by an inhibitor 30 such as Keap1 and a transcription factor 40 such as Nrf2. Now free of the inhibitor 30, the transcription factor 40 can cross over the nuclear membrane 16 to enter the nucleus 14. In other embodiments, the inducer 20 disrupts the complex formed by the inhibitor 30 and the transcription factor 40 by binding to the inhibitor 30 and allowing the transcription factor 40 to be freed. In some cases, once the transcription factor 40 enters the nucleus it can interact with and/or activate one or more response elements 50 such as an Antioxidant Response Element (ARE). The response elements 50 can then interact to promote transcription of endogenous antioxidant genes 60 (e.g., Phase II genes).
In some embodiments, various transcription factors are involved in upregulating one or more endogenous antioxidant systems. For example, transcription factors involved in upregulating one or more endogenous antioxidant systems can include Nrf2. Nrf2 is a transcription factor that is encoded in humans by the NFE2L2 gene and that regulates expression of antioxidants in response to oxidative damage caused by injury and inflammation. As described above, Nrf2 can be maintained in the cytoplasm of the cell under normal conditions and can be degraded fairly quickly. Under oxidative stress conditions, and/or through interaction with an inducer, Nrf2 can translocate to the nucleus to promote transcription of antioxidant genes. In some cases, Nrf2 can promote transcription of various antioxidant genes including Phase II genes, NQO1, GCL, sulfiredoxin 1 (SRXN1) and thioredoxin reductase 1 (TXNRD1), HO-1, GST family genes, and UDP-glucuronosyltransferase (UGT) family genes.
In some embodiments, transcription factors involved in upregulating one or more endogenous antioxidant systems can include an NF-κB complex. In some cases, the NF-κB transcription factor is involved in cellular response to free radicals. NF-κB is often referred to as a rapid-acting primary transcription factor because of its ability to respond quickly to harmful cellular stimuli. NF-κB responds quickly to harmful cellular stimuli by being watchfully present in the cell in an inhibitor-bound inactive state. Once the cell detects harmful cellular stimuli, NF-κB can be quickly activated by degrading the bound inhibitor and freeing NF-κB to translocate to the nucleus to promote transcription of certain genes, including endogenous antioxidant system genes.
In some embodiments, transcription factors involved in upregulating one or more endogenous antioxidant systems can include the PPAR (peroxisome proliferator-activated receptors) family of transcription factors. The PPAR family includes at least the PPARα, PPARβ/δ, and PPARγ transcription factors. Members of the PPAR family are expressed in various tissues with PPARα expressed at least in liver, kidney, heart, muscle, and adipose tissue and with PPARβ/δ expressed at least in brain, adipose tissue, and skin. PPARγ can be expressed in three different forms, γ1, γ2, and γ3, with γ1 expressed in most tissues including heart, muscle, colon, kidney, pancreas, and spleen, with γ2 expressed mainly in adipose tissue, and with γ3 expressed in macrophages, large intestine, and white adipose tissue. In some cases, PPAR transcription factors can bind with certain receptors (e.g. retinoid X receptors) to promote transcription of certain genes, including endogenous antioxidant genes 60 (e.g., Phase II genes).
In some embodiments, upregulating compounds include any compound and/or mixture of compounds suitable for upregulating an endogenous antioxidant system. For example, upregulating compounds can include any compound and/or mixture of compounds that act as an inducer to upregulate an endogenous antioxidant system. In some cases, the upregulating compounds can include any compound that can translocate to the cytoplasm and/or nucleus to upregulate an endogenous antioxidant system. In other cases, the upregulating compounds can include any compound that can interact with one or more signaling molecules and/or signaling complexes to signal upregulation of an endogenous antioxidant system. In yet other cases, the upregulating compounds can include any compound that can directly interact with one or more signaling molecules and/or signaling complexes to signal upregulation of an endogenous antioxidant system. In some cases, the upregulating compounds can include any compound that can indirectly interact with one or more signaling molecules and/or signaling complexes to signal upregulation of an endogenous antioxidant system. In other cases, the upregulating compounds can include a nutrient, an herbal supplement, a plant extract, or any other similar compound.
In some embodiments, upregulating compounds comprise one or more of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. For example, the upregulating compounds can include lipoic acid. In some cases, lipoic acid can include one or more of alpha lipoic acid (ALA), racemic alpha lipoic acid, di-hydro alpha lipoic acid, R-(+) alpha lipoic acid, S-(−) alpha lipoic acid, R-(+) dihydro alpha lipoic acid, S-(−) dihydro alpha lipoic acid, metal salts thereof, esters thereof, or combinations thereof. FIG. 2A shows one chemical formula of alpha lipoic acid.
In some embodiments, the upregulating compounds comprise resveratrol or a similar stilbenoid. Resveratrol can include one or more of 3,5,4′-trihydroxy-trans-stilbene, 3,4′,5-Stilbenetriol, trans-Resveratrol, (E)-5-(p-Hydroxystyryl)resorcinol, and (E)-5-(4-hydroxystyryl)benzene-1,3-diol. Resveratrol can include the cis-(Z) and/or trans-(E) isomers. Resveratrol can be derived from any suitable source including plant sources such as grapes or the skin of grapes, seeds of muscadine grapes, blueberries, raspberries, mulberries, bilberries, peanuts, Japanese knotweed, and cocoa powder. FIG. 2B shows one chemical formula of resveratrol.
In some embodiments, the upregulating compounds comprise curcumin. In other embodiments, curcumin comprises one or more of curcumin and any other suitable curcuminoid, Curcumin can include any suitable tautomeric form of curcumin including, but not limited to, a 1,3-diketo form or an enol form. Curcumin can also include any suitable turmeric extract (e.g., desmethoxycurcumin and/or bis-desmethoxycurcumin. FIG. 2C shows one chemical formula of curcumin.
In some embodiments, the upregulating compounds comprise epigallocatechin gallate (EGCG). In other embodiments, the upregulating compounds comprise any suitable ester of epigallocatechin and gallic acid. EGCG can also include one or more of [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)chroman-3-yl] 3,4,5-trihydroxybenzoate, (2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3- yl 3,4,5-trihydroxybenzoate, and (−)-Epigallocatechin gallate. EGCG can be derived from any suitable source, include plant sources such as the leaves of white tea, the leaves of green tea, the leaves of black tea, apple skin, plums, onions, hazelnut, pecans, and carob. FIG. 3A shows one chemical formula of EGCG.
In some embodiments, the upregulating compounds comprise rutin. In other embodiments, rutin comprises one or more of rutoside, quercetin-3-O-rutinoside, phytomelin, birutan, Eldrin, birutan forte, rutin trihydrate, globularicitrin, violaquercetin, and sophorin. Rutin can also include 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-{[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-({[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxy}methyl)oxan-2-yl]oxy}-4H-chromen-4-one and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyloxy]-4H-chromen-4-one. Rutin can be derived from any suitable source, include plant sources such as Carpobrotus edulis, Ruta graveolens, buckwheat, asparagus, fruit of the fava d'anata tree, fruits and flowers of the pagoda tree, oranges, grapefruits, lemons, limes, mulberry fruit, ash tree fruits, aronia berries, cranberries, and peaches. FIG. 3B shows one chemical formula of rutin.
In some embodiments, the upregulating compounds comprise Olivol®. In some cases, Olivol® can comprise any prepared extract of olive fruit. In other cases, Olivol® can comprise an extract of olive fruit prepared by methods disclosed in U.S. Pat. No. 6,358,542 and/or U.S. Pat. No. 6,361,803, the disclosures of which are hereby incorporated by reference. In yet other cases, Olivol® can be prepared by providing olive pulp by-product of olive oil production, extracting the pulp with a polar aqueous solvent to form an aqueous phase, passing the aqueous phase through a polymeric resin to trap antioxidants on the resin, washing the polymeric resin with polar organic solvent to release antioxidants from the resin to produce a solution of antioxidants in the polar organic solvent. Olivol® can comprise a phenolic antioxidant mixture of tyrosol, hydroxytyrosol, verbacoside, and other related compounds.
In some embodiments, the upregulating compounds comprise quercetin. In some cases quercetin can include 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. Quercetin can also include sophoretin, meletin, quercetine, xanthaurine, quercetol, quercitin, quertine, and flavin meletin. Quercetin can be derived from any suitable source including plant sources such as capers, radish leaves, carob fiber, dill, cilantro, Hungarian wax pepper, fennel leaves, red onion, radicchio, watercress, buckwheat, kale, chokeberry, cranberry, lingonberry, black plums, cow peas, sweet potato, blueberry, sea buckthorn berry, rowanberry, crowberry, prickly pear cactus fruits, red delicious apples, broccoli, bilberry, black tea, and green tea. FIG. 3C shows one chemical formula of quercetin.
In some embodiments, the upregulating compounds comprise hesperetin. In some cases, hesperetin can include (S)-2,3-Dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1-benzopyran-4-one. In other cases, hesperetin can include one or more of hesperidin, hesperetin 7-rutinoside, neohesperidin, 7-neohesperidoside, and hesperetin 7-rhamnoside. Hesperidin can be derived from any suitable source, including plant sources such as citrus fruits. FIG. 4 shows one chemical formula of hesperetin.
In some embodiments, the methods include methods of preparing nutritional supplements and compositions of nutritional supplements that comprise one or more of an upregulating compound mixture, an exogenous antioxidant mixture, and a mineral mixture. In other embodiments, the methods of preparing nutritional supplements and compositions of nutritional supplements comprise preparing nutritional supplements that comprise an upregulating compound mixture and an exogenous antioxidant mixture in a first part and a mineral mixture in a second part. In yet other embodiments, the methods of preparing nutritional supplements and compositions of nutritional supplements comprise preparing nutritional supplements that comprise an upregulating compound mixture in a first part, an exogenous antioxidant mixture in a second part, and a mineral mixture in a third part. In some embodiments, the upregulating compound mixture in a first part and the exogenous antioxidant mixture in a second part is combined in a single first vehicle and the mineral mixture in a third part is prepared as a single second vehicle.
In some embodiments, the upregulating compound mixture, the exogenous antioxidant mixture, and the mineral mixture are combined in the form of a single bilayer tablet or capsule. In these embodiments, the upregulating compound mixture and the exogenous antioxidant mixture are contained within a first part of the tablet or capsule and the mineral mixture is contained in a second part of the tablet or capsule. The first part and the second part can be maintained partially or completely separated from each other using any known separation technique. For example, these separation techniques can include forming the first part as a homogeneous first layer in the tablet and the second part as a homogeneous second layer in the table. The contact between the first layer and the second layer is minimized because they only contact each other at the interface between the first and second layers. In other embodiments, the separation technique includes using one or more of a coating, a film, and an inert layer to separate the first layer and second layers.
In some embodiments, a typical tablet shape comprises a caplet which has about the shape of a rectangular box. A bi-layer tablet in these configurations can comprise two or more of these boxes sandwiched together, with each box comprising a layer. An amount of material that is in contact at an interface between the layers can be estimated from an amount of material required to coat the entire tablet. The estimation is carried out by determining the amount of material required to coat the entire tablet and approximating that about half of this amount is an amount needed for the interface between the layers. Because the amount of material required to coat the entire tablet can range from about 1 to about 5% of the mass of the entire tablet, half of this amount can be approximated to range between about 0.5% and about 2.5% of the mass of the entire tablet. Therefore, about 0.5% and about 2.5% of the mass of the entire tablet can be approximated as the amount of material that is in contact at an interface between the layers.
In some embodiments, this separation technique includes forming the first part as a first layer in the tablet and the second part as a second layer in the tablet. Both the first and second layers are formed with a concentration gradient where one or more of the active ingredients in the bi-layer tablet is concentrated at an exterior of the tablet and minimized at the location at the interface where the two layers contact each other. In these embodiments, contact between the first layer and the second layer is limited to the interface between the first and second layers.
In some embodiments, this separation technique includes forming the first part as a first layer in the tablet and the second part as a second layer in the tablet. In these embodiments, the contact between the first layer and the second layer is reduced by providing a barrier between the two layers. In some configurations, the barrier can comprise a physical barrier, such as a film of the same material as the capsule that dissolves on contacting saliva. The physical barrier can have any thickness sufficient to prevent and/or reduce any contact between the two layers. In other configurations, the physical barrier can comprise a chemical component that prevents the two layers from reacting with each other. Examples of such chemical components include magnesium carbonate, potassium carbonate and sodium carbonate.
In other embodiments, the first layer is prepared as a first powder and the second layer is prepared as a second powder. In some cases, the first powder and the second powder can be combined. While the first and second portions can be mixed, the contact between the two ingredients can be minimized or eliminated by coating the first and/or second powders with a non-reactive layer having a thickness sufficient to prevent any substantial contact and/or reaction between the two ingredients. Examples of non-reactive layers include one or more of cellulose and food grade wax.
In some embodiments, the first layer is prepared as a first liquid and the second layer is prepared as a second liquid. A capsule can be prepared that contains a first, inner capsule containing one of these two liquids. The first capsule can be completely contained within a second, outer capsule that contains the other liquid. Thus, the two liquids are kept separated from each other by the inner capsule.
In some embodiments, the upregulating compound mixture and the exogenous antioxidant mixture are formulated as a single vehicle (e.g., a single tablet, dosage, or aliquot). While, the upregulating compound mixture can include any suitable upregulating compound, in some embodiments, the upregulating compound mixture includes one or more of the upregulating compounds described above (e.g., alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin). In other embodiments, the upregulating compound mixture includes one or more bioflavonoids (e.g., sulforaphane precursors found in broccoli extracts, sulforaphane, glucoraffnin, and other suitable bioflavonoids). In yet other embodiments, the upregulating compound mixture comprises the compounds at the concentrations (e.g., mg of active ingredient (AI) and % by weight in mixture) as described in Table 1.

TABLE 1

		% by weight
Ingredient	mg of AI	in mixture

Alpha Lipoic Acid	25	22
Resveratrol	10	9
Curcumin Phytosome Complex	18	16
(Meriva- Bioavailable curcuminoids
containing 3.25 mg curcuminoids)
Green Tea Extract (standardized to	17.5	15
EGCG)
Olivol ® (Olive Fruit Extract)	7.5	7
Rutin	10	9
Quercetin	15	13
Hesperidin	10	9

In some embodiments, alpha lipoic acid comprises between about 15 mg to about 35 mg of AI of the upregulating compound mixture. In other embodiments, alpha lipoic acid comprises between about 20 mg to about 30 mg of AI of the upregulating compound mixture. In yet other embodiments, alpha lipoic acid comprises up to about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg of AI, or any intermediary value thereof, of the regulating compound mixture.
In some embodiments, alpha lipoic acid comprises between about 15% to about 35% by weight of the upregulating compound mixture. In other embodiments, alpha lipoic acid comprises between about 20% to about 25% by weight of the upregulating compound mixture. In yet other embodiments, alpha lipoic acid comprises up to about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% by weight of the upregulating mixture, or any intermediary value thereof of the regulating compound mixture.
In some embodiments, resveratrol comprises between about 1 mg to about 25 mg of AI of the upregulating compound mixture. In other embodiments, resveratrol comprises between about 5 mg to about 15 mg of AI of the upregulating compound mixture. In yet other embodiments, resveratrol comprises up to about 1 mg, about 2 mg, about 5 mg, about 8 mg, about 9 mg, about 10 mg, about 12 mg, about 15 mg, or any intermediary value thereof of AI of the regulating compound mixture.
In some embodiments, resveratrol comprises between about 1% to about 25% by weight of the upregulating compound mixture. In other embodiments, resveratrol comprises between about 5% to about 15% by weight of the upregulating compound mixture. In yet other embodiments, resveratrol comprises up to about 1%, about 2%, about 5%, about 9%, about 10%, about 12%, about 15% by weight, or any intermediary value thereof, of the regulating compound mixture. In some embodiments, curcumin comprises between about 10 mg to about 35 mg of AI of the upregulating compound mixture. In other embodiments, curcumin comprises between about 15 mg to about 25 mg of AI of the upregulating compound mixture. In yet other embodiments, curcumin comprises up to about 5 mg, about 10 mg, about 15 mg, about 18 mg, about 20 mg, about 22 mg, about 35 mg, about 30 mg, or any intermediary value thereof of AI of the regulating compound mixture.
In some embodiments, curcumin comprises between about 10% to about 35% by weight of the upregulating compound mixture. In other embodiments, curcumin comprises between about 15% to about 25% by weight of the upregulating compound mixture. In yet other embodiments, curcumin comprises up to about 5%, about 10%, about 15%, about 16%, about 20%, about 22%, about 25%, about 30%, about 35% by weight or any intermediary value thereof, of the regulating compound mixture.
In some embodiments, EGCG comprises between about 10 mg to about 35 mg of AI of the upregulating compound mixture. In other embodiments, EGCG comprises between about 15 mg to about 25 mg of AI of the upregulating compound mixture. In yet other embodiments, EGCG comprises up to about 5 mg, about 10 mg, about 15 mg, about 18 mg, about 20 mg, about 22 mg, about 35 mg, about 30 mg, or any intermediary value thereof of AI of the regulating compound mixture.
In some embodiments, EGCG comprises between about 10% to about 35% by weight of the upregulating compound mixture. In other embodiments, EGCG comprises between about 15% to about 25% by weight of the upregulating compound mixture. In yet other embodiments, EGCG comprises up to about 5%, about 10%, about 15%, about 16%, about 20%, about 22%, about 25%, about 30%, about 35% by weight or any intermediary value thereof, of the regulating compound mixture.
In some embodiments, Olivol® comprises between about 1 mg to about 20 mg of AI of the upregulating compound mixture. In other embodiments, Olivol® comprises between about 5 mg to about 15 mg of AI of the upregulating compound mixture. In yet other embodiments, Olivol® comprises up to about 1 mg, about 2 mg, about 5 mg, about 7.5 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, or any intermediary value thereof, of AI of the regulating compound mixture.
In some embodiments, Olivol® comprises between about 1% to about 15% by weight of the upregulating compound mixture. In other embodiments, Olivol® comprises between about 5% to about 12% by weight of the upregulating compound mixture. In yet other embodiments, Olivol® comprises up to about 1%, about 2%, about 5%, about 7%, about 8%, about 10%, about 12%, about 15%, about 18% by weight or any intermediary value thereof, of the regulating compound mixture.
In some embodiments, rutin comprises between about 1 mg to about 30 mg of AI of the upregulating compound mixture. In other embodiments, rutin comprises between about 5 mg to about 15 mg of AI of the upregulating compound mixture. In yet other embodiments, rutin comprises up to about 1 mg, about 2 mg, about 5 mg, about 8 mg, about 10 mg, about 12 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, or any intermediary value thereof, of AI of the regulating compound mixture.
In some embodiments, rutin comprises between about 1% to about 30% by weight of the upregulating compound mixture. In other embodiments, rutin comprises between about 5% to about 25% by weight of the upregulating compound mixture. In yet other embodiments, rutin comprises up to about 5%, about 10%, about 13%, about 15%, about 18%, about 20%, about 22%, about 25%, about 30% by weight or any intermediary value thereof, of the regulating compound mixture.
In some embodiments, quercetin comprises between about 1 mg to about 30 mg of AI of the upregulating compound mixture. In other embodiments, quercetin comprises between about 10 mg to about 20 mg of AI of the upregulating compound mixture. In yet other embodiments, quercetin comprises up to about 1 mg, about 5 mg, about 10 mg, about 12 mg, about 15 mg, about 18 mg, about 20 mg, about 22 mg, about 25 mg, about 30 mg, or any intermediary value thereof, of AI of the regulating compound mixture.
In some embodiments, quercetin comprises between about 1% to about 30% by weight of the upregulating compound mixture. In other embodiments, quercetin comprises between about 5% to about 25% by weight of the upregulating compound mixture. In yet other embodiments, quercetin comprises up to about 5%, about 10%, about 13%, about 15%, about 18%, about 20%, about 22%, about 25%, about 30% by weight or any intermediary value thereof, of the regulating compound mixture.
While the exogenous antioxidant mixture can comprise any suitable exogenous antioxidant, at least in some embodiments the exogenous antioxidant mixture comprises one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene. In other embodiments, the exogenous antioxidant mixture comprises the compounds at the concentrations (e.g., mg of active ingredient (AI) and international unit (IU)) as described in Table 2.

TABLE 2

Ingredient	mg of AI	IU

Mixed Carotenoids *	0.1	100 IU
Beta carotene	1.29	2150 IU
Retinyl Acetate	0.258	750 IU
Vitamin C (Poly C) **	100
Vitamin D3 (Cholecalciferol)	0.0125	500 IU
Vitamin E (d-alpha-tocopheryl succ.)	41.3	50 IU
Mixed Tocopherols	20
Vitamin K1	0.12
Vitamin K2 (menaquinone, MK-7)	0.015
Vitamin B1 (thiamin HCL)	7.5
Vitamin B2 (riboflavin)	7.5
Niacin	2.5
Niacinamide	7.5
Vitamin B6 (pyridoxine HCL)	8
Folic Acid	0.15
Vitamin B12 (methylcobalamin)	0.05
Biotin	0.075
Pantothenic Acid	22.5
Inositol	32
Choline bitartrate	62.5
Coenzyme Q-10	3
Lutein	0.15
Lycopene	0.25

* Mixed carotenoids comprises a mixture of alpha-carotene, beta-carotene, gamma-carotene, and lycopene
** Vitamin (Poly C) was a mixture of calcium ascorbate, potassium ascorbate, magnesium ascorbate, and zinc ascorbate.

In some embodiments, the exogenous antioxidant mixture comprises individual exogenous antioxidant compounds at any suitable concentration. For example, mixed carotenoids can comprise between about 0.01 and 1 mg of AI or between 1 and about 200 IU, beta carotene can comprise between about 0.01 and 3 mg of AI or between 1000 and about 3000 IU, retinyl acetate can comprise between about 0.01 and 1 mg of AI or between 100 and about 1500 IU, vitamin C can comprise between about 10 and 200 mg of AI, vitamin D3 can comprise between about 0.001 and 1 mg of AI or between 100 and about 1000 IU, vitamin E can comprise between about 10 and 100 mg of AI or between 10 and about 150 IU, mixed tocopherols can comprise between about 1 and 50 mg of AI, vitamin K1 can comprise between about 0.01 and 1 mg of AI, vitamin K2 can comprise between about 0.0001 and 1 mg of AI, vitamin B1 can comprise between about 1 and 20 mg of AI, vitamin B2 can comprise between about 1 and 20 mg of AI, niacin can comprise between about 1 and 20 mg of AI, niacinamide can comprise between about 1 and 20 mg of AI, vitamin B6 can comprise between about 1 and 20 mg of AI, folic acid can comprise between about 0.01 and 2 mg of AI, vitamin B12 can comprise between about 0.001 and 2 mg of AI, biotin can comprise between about 0.001 and 2 mg of AI, pantothenic acid can comprise between about 1 and 50 mg of AI, inositol can comprise between about 1 and 100 mg of AI, choline bitartrate can comprise between about 1 and 200 mg of AI, coenzyme Q-10 can comprise between about 0.1 and 20 mg of AI, lutein can comprise between about 0.01 and 2 mg of AI, and lycopene can comprise between about 0.01 and 2 mg of AI.
While the mineral mixture can comprise any suitable exogenous antioxidant, at least in some embodiments the mineral mixture comprises one or more of calcium, calcium citrate, calcium ascorbate, magnesium, magnesium citrate, magnesium ascorbate, iodine, potassium iodine, zinc, zinc citrate, selenium, L-selenomethionine, sodium selenite, copper, copper gluconate, manganese, manganese gluconate, chromium, chromium polynicotinate, molybdenum, molybdenum citrate, boron, boron citrate, silicon, calcium silicate, vanadium, vanadium citrate, ultra-trace minerals, and N-acetyl-L-cysteine. In other embodiments, the mineral mixture comprises individual mineral compounds at the concentrations (e.g., mg of active ingredient (AI)) as described in Table 3.

	TABLE 3

	Ingredient	mg of AI

	Total Calcium	56.25
	Calcium Citrate	48.25
	Calcium Ascorbate ^1,3	8
	Magnesium (citrate)⁴	50.13
	Magnesium Ascorbate^2,3,4	6.12
	Iodine (potassium iodide)	0.125
	Zinc (citrate)	5.0
	Selenium (L-selenomethionine)	0.0275
	Selenium (sodium selenite)	0.0225
	Copper (gluconate)	0.5
	Manganese (gluconate)	0.5
	Chromium (polynicotinate)	0.075
	Molybdenum (citrate)	0.0125
	Boron (citrate)	0.75
	Silicon (calcium silicate)	1
	Vanadium (citrate)	0.01
	Ultra-trace Minerals	0.75
	N-acetyl L-cysteine	45

	¹Adds an equivalent of 70.9 mg of AI of vitamin C from calcium ascorbate
	²Adds an equivalent of 79.1 mg of AI of vitamin C from magnesium ascorbate
	³Total vitamin C equivalent equals 150 mg of AI
	⁴Total magnesium content equals 56.25 mg of AI

In some embodiments, the mineral mixture is configured to provide one or more cofactors related to upregulating endogenous antioxidant activity. For example, the mineral mixture can provide one or metal ions that act as a cofactor for an antioxidant enzyme and/or one or more endogenous antioxidant systems.
In some embodiments, the mineral mixture comprises individual mineral compounds at any suitable concentration. For example total calcium can comprise between about 10 and about 200 mg of AI, calcium citrate can comprise between about 1 and about 200 mg of AI, calcium ascorbate can comprise between about 1 and about 200 mg of AI, magnesium citrate can comprise between about 1 and about 200 mg of AI, magnesium ascorbate can comprise between about 0.1 and about 20 mg of AI, potassium iodide can comprise between about 0.001 and about 10 mg of AI, zinc citrate can comprise between about 0.1 and about 50 mg of AI, L-selenomethionine can comprise between about 0.001 and about 1 mg of AI, sodium selenite can comprise between about 0.001 and about 1 mg of AI, copper gluconate can comprise between about 0.01 and about 10 mg of AI, manganese gluconate can comprise between about 0.01 and about 10 mg of AI, chromium polynicotinate can comprise between about 0.001 and about 1 mg of AI, molybdenum citrate can comprise between about 0.001 and about 1 mg of AI, boron citrate can comprise between about 0.01 and about 10 mg of AI, calcium silicate can comprise between about 0.1 and about 10 mg of AI, vanadium citrate can comprise between about 0.001 and about 1 mg of AI, ultra-trace minerals can comprise between about 0.01 and about 10 mg of AI, and N-acetyl-L-cysteine can comprise between about 1 and about 100 mg of AI.
In some embodiments, one or more of the upregulating compound mixture, the exogenous antioxidant compound mixtures and the mineral mixture is prepared as a solid formulation. For example, the upregulating compound mixture and the exogenous antioxidant mixture are formulated as a single solid vehicle (e.g., a solid tablet) and the mineral mixture is formulated as a separate single solid vehicle (e.g., a solid tablet). In other embodiments, one or more of the upregulating compound mixture, the exogenous antioxidant compound mixtures and the mineral mixture is prepared as a liquid formulation (e.g., a liquid capsule). In some cases, the upregulating compound mixture and the exogenous antioxidant mixture are formulated as a single liquid vehicle (e.g., a liquid capsule) and the mineral mixture is formulated as a separate single liquid vehicle (e.g., a liquid capsule). In yet other embodiments, one or more of the upregulating compound mixture, the exogenous antioxidant compound mixtures and the mineral mixture is prepared as a solid granular formulation. For example, the upregulating compound mixture and the exogenous antioxidant mixture are formulated as solid granular vehicle (e.g., a granular filled capsule) and the mineral mixture is formulated as a separate solid granular vehicle (e.g., a granular filled capsule).
In some embodiments, one or more of the upregulating compound mixture, the exogenous antioxidant compound mixtures and the mineral mixture comprise any suitable additive. For example, suitable additive can include binders, disintegrants, lubricants, flowing agents, flavorings, coatings, and any combination thereof. In some cases, binders can include microcrystalline cellulose, modified cellulose (e.g., Klucel), pre-gelatinized starch, or combinations thereof. In other cases, disintegrants can include croscarmellose sodium. Lubricant can include ascorbyl palmitate, vegetable fatty acid, or combinations thereof. Flowing agents can include silicon dioxide. Flavorings can include vanilla extract. In other embodiments, additive may include one or more of maltodextrin, organic maltodextrin, lecithin, sunflower lecithin, palm olein, organic palm olein, guar gum, and organic guar gum. In yet other embodiments, additives comprise any suitable amount of the nutritional supplement.
The nutritional supplement can be prepared in any suitable form, including but not limited to, tablets, capsules, and powders. Solid diluents or carriers for the solid forms can be lipids, carbohydrates, proteins, mineral solids (e.g., starch, sucrose, kaolin, dicalcium phosphate, gelatin, acacia, corn syrup, corn starch, talc, and their combinations), and combinations thereof. Capsules can be formulated with known diluents and excipients, for example, edible oils, talc, calcium carbonate, calcium stearate, magnesium stearate, and combinations thereof. Liquid preparations for oral administration may be prepared in water or aqueous solutions which advantageously contain suspending agents, such as for example, sodium carboxymethylcellulose, methylcellulose, acacia, polyvinyl pyrrolidone, polyvinyl alcohol and combinations thereof.
In some embodiments, the nutritional supplements comprise preservatives in the nature of bactericidal and fungicidal agents including, but not limited to, parabens, chlorobutanol, benzyl alcohol, phenol, thimerosal, and the like. In some cases, the nutritional supplements can comprise isotonic agents such as sugars or sodium chloride. Carriers and vehicles include vegetable oils, water, ethanol, and polyols, for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like.
The nutritional supplements can be prepared using any known method that will manufacture the desired form with the components in the desired concentrations. In some embodiments, the ingredients for one of the upregulating compound mixture, the exogenous antioxidant mixture, or mineral mixture are first weighed out and then transferred to a blender to be mixed. After the respective ingredients have been mixed in the blender, they are transferred to a hopper that feeds a tablet press that forms compressed tablets. The compressed tablets can be transferred to a coating pan where the coating solution is applied and the tablets are dried. The same process can be repeated for the remaining mixtures.
In some embodiments, the nutritional supplement is administered to a human or an animal. While the nutritional supplement can be administered in any suitable manner, at least in some embodiments, the nutritional supplement is configured to be ingested by the human or the animal. In other embodiments, the method of administration can be adapted to the form of the nutritional supplement. For example, the nutritional supplement can be configured in the form of a tablet and/or capsule that can be swallowed by a human or an animal. In some cases, the nutritional supplement can be configured as a powder and/or a granular solid that can be added to a food or a beverage that can be consumed by the human or the animal. In other cases, the nutritional supplement can be configured as a liquid that is encapsulated in a gel capsule that can be swallowed or otherwise ingested. In yet other cases, the nutritional supplement can be configured as a liquid that is swallowed or otherwise ingested. In some cases, the nutritional supplement can be configured in a chewable form, such as a gelatin-based chewable dose.
In some embodiments, the nutritional supplement is administered in any suitable dosage. In other embodiments, the dosage of the nutritional supplement is modified based on one or more of an individual's weight, height, age, gender, pregnancy status, breastfeeding status, metabolism, health status, ethnicity, genetics, environment, diet, fitness level, cardiac health, body mass index, and/or lifestyle. In some embodiments, the dosage of the upregulating compound mixture is a daily dose of between about 1 and 6 times the amount listed in Table 1. In other embodiments, the dosage of the upregulating compound mixture is a daily dosage of about 1, 2, 3, 4, 5, or 6 times the amount listed in Table 1. In yet other embodiments, the dosage of the upregulating compound mixture is about 4 times the amount listed in Table 1 for an adult. In some embodiments, the dosage of the upregulating compound mixture is about 2-3 times the amount listed in Table 1 for an adolescent. In other embodiments, the dosage of the upregulating compound mixture is about 0.5 to 1 times the amount listed in Table 1 for a child.
In some embodiments, the dosage of the endogenous antioxidant compound mixture is a daily dose of between about 1 and 6 times the amount listed in Table 2. In other embodiments, the dosage of the endogenous antioxidant compound mixture is a daily dosage of about 1, 2, 3, 4, 5, or 6 times the amount listed in Table 2. In yet other embodiments, the dosage of the endogenous antioxidant compound mixture is about 4 times the amount listed in Table 2 for an adult. In some embodiments, the dosage of the endogenous antioxidant compound mixture is about 2-3 times the amount listed in Table 2 for an adolescent. In other embodiments, the dosage of the endogenous antioxidant compound mixture is about 0.5 to 1 times the amount listed in Table 2 for a child.
In some embodiments, the dosage of the mineral compound mixture is a daily dose of between about 1 and 6 times the amount listed in Table 3. In other embodiments, the dosage of the mineral compound mixture is a daily dosage of about 1, 2, 3, 4, 5, or 6 times the amount listed in Table 3. In yet other embodiments, the dosage of the mineral compound mixture is about 4 times the amount listed in Table 3 for an adult. In some embodiments, the dosage of the mineral compound mixture is about 2-3 times the amount listed in Table 3 for an adolescent. In other embodiments, the dosage of the mineral compound mixture is about 0.5 to 1 times the amount listed in Table 3 for a child.
In some embodiments, the nutritional supplement is administered as a single daily dose. In other embodiments, the nutritional supplement is administered as multiple doses within a set period of time (e.g., a 24 hour period of time). In yet other embodiments, a single dose is divided into aliquots that are administered within a set period of time (e.g., a 24 hour period of time). In some embodiments, the nutritional supplement is administered as a single weekly dose. In other embodiments, the nutritional supplement is administered as a single monthly dose.
In some embodiments, the nutritional supplement is daily administered over a period of days. In other embodiments, the nutritional supplement is administered daily over a period of weeks. In yet other embodiments, the nutritional supplement is administered daily over a period of years.
In some embodiments, the nutritional supplement is administered to a human or an animal to reduce and/or prevent free radical damage by synergistically upregulating endogenous antioxidant systems, providing exogenous antioxidants, and providing minerals. In other embodiments, the nutritional supplement is administered to a human or an animal to upregulate endogenous antioxidant systems within the human or animal. In yet other embodiments, the nutritional supplement is administered to a human or an animal to upregulate endogenous antioxidant systems within the human or animal to reduce and/or prevent free radical damage. In some embodiments, the nutritional supplement is administered to a human or an animal to reduce and/or prevent damage by free radicals generated during oxidative phosphorylation. For example, the nutritional supplements can be administered to upregulate Phase II genes to reduce and/or prevent free radical damage. The nutritional supplements can also be administered to activate a transcription factor such as Nrf2, NF-κB, PPARα, PPARβ/δ, and/or PPARγ. The nutritional supplements can also be administered to promote transcription of endogenous antioxidant gene such as NQO1, GCL, sulfiredoxin 1 (SRXN1) and thioredoxin reductase 1 (TXNRD1), HO-1, GST family genes, and UDP-glucuronosyltransferase (UGT) family genes.

Example 1

Various receptor assays were carried out for test compounds corresponding to ingredients of the nutritional supplement composition. In general, the receptor assays utilized reporter cells that either expressed a native receptor or a receptor hybrid. The receptor hybrids were engineered so that the native N-terminal DNA binding domain (DBD) was replaced with a yeast Gal4 DBD. The reporter cells expressed a hybrid receptor comprising either the native receptor (Nrf2 and NF-κB) or the N-terminal Gal4 DNA binding domain fused to the ligand binding domain of the specific human nuclear receptor (PPARα, PPARδ, and PPARγ). The reporter gene (e.g., firefly luciferase) was functionally linked to either upstream receptor-specific response elements (GRE) or the Gal4 upstream activation sequence (UAS). A summary of the receptors, the reporter cells used for each particular receptor assay, and the reference compounds used to confirm performance of the receptor assays are indicated below in Table 4.

TABLE 4

					Refer-
Receptor			Host		ence
(gene	Recep-	Reporter	Cell	Reference	Antag-
symbol)	tor form	Vector	Line	Agonist	onist

PPARα	Gal4 DBD	Gal4 UAS-	CHO	GW590735	np
(NR1C1)	hybrid	Luciferase
	receptor
PPARδ	Gal4 DBD	Gal4 UAS-	CHO	GW0742	np
(NR1C2)	hybrid	Luciferase
	receptor
PPARγ	Gal4 DBD	Gal4 UAS-	CHO	Rosiglita-	np
(NR1C3)	hybrid	Luciferase		zone
	receptor
Nrf2	Native	ARE-	CV1	L-	np
	Receptor	Luciferase		Sulforophane
NF-κB	Native	NF-κB	HEK293	Phorbol ester	na
	NF-κB	GRE-		(PMA)
		Luciferase

np = assay not performed
na = not available
CHO = Chinese hamster ovary cell line
HEK293 = human embryonic kidney 293 cell line
CV1 = mammalian CV1 cell line

The test compounds included alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, hesperetin, and a mixture of the all of the test compounds. A summary of the test compounds are shown below in Table 5.

TABLE 5

						Effective
Test	Raw		Actual mass		Actual molar	molar
Compound	Material	Purity	concentration	MW	concentration	concentration

Alpha	155 mg/ml	74%	114.8 mg/ml	206.32	550 mM	550 μM
lipoic acid
Resveratrol	16 mg/ml	47.6%	7.6 mg/ml	228.25	33.3 mM	3.7 μM
Curcumin	2.5	100%	2.5 mg/ml	368.69	6.79 mM	n/a
EGCG	20	40%	8 mg/ml	458.372	17.5 mM	n/a
Olivol ®	20	100%	20 mg/ml	154	129 mM	n/a
Rutin	30	100%	30 mg/ml	610.50	49.1 mM	n/a
Quercetin	68	100%	68 mg/ml	302.2	22.5 mM	7.5 μM
Hesperetin	2.5	35%	0.875 mg/ml	610.50	1.43 mM	n/a
Mixture	7.41	n/a	n/a	n/a	n/a	n/a

The test compounds were assayed for activity against human PPARα, PPARδ, PPARγ, and Nrf2 in agonist mode. For the agonist assays, separate suspensions of each of the PPARα, PPARδ, PPARγ, Nrf2 reporter cells were prepared in cell recovery medium containing 10% charcoal stripped fetal bovine serum. Next, 100 μL aliquots of the PPARα reporter cells were dispensed into each test well of a white 96-well assay plate. Assay plates with PPARδ, PPARγ, and Nrf2 reporter cells were prepared in similar fashion. Dilutions of the test compounds were serially diluted using compound screening medium containing 10% charcoal stripped fetal bovine serum to generate 2×-concentration test compound samples. Control solutions of known agonists of the each of the PPARα, PPARδ, PPARγ, and Nrf2 receptors were prepared along with a vehicle control. 100 μL aliquots of the 2×-concentration test compound samples, control solutions, and vehicle control were dispensed into separate test wells of each white 96-well assay plate in triplicate. The assay plates were incubated at 37° C. for 24 h. After incubation, media was removed from each test well while leaving behind the receptor cells and 100 μL of luciferase detection reagent was added to each test well and emitted light from each test well of the assay plates was detected. The emitted light from each test well was recorded as relative light units (RLU).
The test compounds were assayed for activity against human NF-κB in antagonist mode. For the agonist assays, a suspension of NF-κB reporter cells were prepared in cell recovery medium containing 10% charcoal stripped fetal bovine serum. Next, 100 μL aliquots of the NF-κB reporter cells were dispensed into each test well of a white 96-well assay plate. Dilutions of the test compounds were serially diluted using compound screening medium containing 10% charcoal stripped fetal bovine serum to generate 2×-concentration test compound samples. A vehicle control was prepared. 100 μL aliquots of the 2×-concentration test compound samples and vehicle control were dispensed into separate test wells of the white 96-well assay plate in triplicate. The assay plate was incubated at 37° C. for 24 h. After incubation, media was removed from each test well while leaving behind the receptor cells. The receptor cells were rinsed once with live cell multiplex buffer, live cell multiplex substrate added, and the plate incubated at 37° C. for 30 minutes. After incubation, fluorescence was measured to determine relative number of live cell per test well. The live cell multiplex substrate was then removed and discarded and 100 μL luciferase detection reagent was added to each test well and emitted light from each test well of the assay plate was detected. The emitted light from each test well was recorded as relative light units (RLU).
The recorded RLU for each test well was correlated to the respective nuclear receptor activities by using the RLU of each dilution of each test compound, the RLU of the control solutions of known agonists, and the RLU of the vehicle controls. The fold-activation was determined for the agonist assays and the percent inhibition and percent live cells were determined for the antagonist assays. The fold-activation for the agonist assays for each serial dilution of each test compound is shown below in Table 6.

TABLE 6

	Active	PPARα	PPARδ	PPARγ	Nrf2
	Ingredient	Fold-	Fold-	Fold-	Fold-
Test	concentration	Activa-	Activa-	Activa-	Activa-
Compound	in μg/ml	tion	tion	tion	tion

Vehicle (DMSO)	0.10%	1.0	1.0	1.0	1.0
Alpha lipoic	0.47	1.2	1.4	0.85	1.0
acid	1.42	1.4	1.5	1.1	1.4
	4.25	1.3	1.1	1.1	1.4
	12.7	1.2	0.80	1.3	1.5
	38.23	1.5	0.52	1.4	1.5
	114.70	2.3	0.29	3.4	2.4
Resveratrol	0.031	1.3	1.6	1.1	1.5
	0.094	1.3	1.1	1.0	1.2
	0.28	1.2	1.4	0.95	1.2
	0.85	1.7	1.5	1.3	4.9
	2.5	1.8	2.1	3.1	4.0
	7.62	1.3	1.5	7.3	3.3
Curcumin	0.007	1.3	1.2	1.0	1.6
	0.022	1.3	1.2	1.0	1.7
	0.067	1.3	1.0	0.95	1.4
	0.20	1.1	1.2	0.96	0.96
	0.60	1.0	1.3	0.90	1.0
	1.81	0.86	1.0	1.1	1.6
EGCG	0.033	1.4	1.3	1.1	1.4
	0.098	1.3	1.4	1.0	1.3
	0.30	1.4	1.2	1.0	1.2
	0.89	1.2	1.3	0.95	1.2
	2.67	1.1	1.1	0.85	1.3
	8	0.85	0.48	0.53	0.74
Olivol ®	0.8	1.4	0.90	0.78	1.0
	0.25	1.5	1.6	1.0	1.4
	0.74	1.3	1.1	1.1	1.4
	2.2	1.5	1.6	1.1	1.4
	6.7	1.4	1.5	1.0	1.4
	20	1.0	0.84	1.0	1.3
Rutin	0.12	1.1	0.95	0.91	1.4
	0.37	1.1	1.0	0.78	1.1
	1.11	0.94	1.3	0.86	0.85
	3.3	0.99	1.1	1.0	1.4
	10.0	1.0	1.3	1.2	1.4
	30	1.0	1.2	1.3	1.5
Quercetin	0.28	1.5	1.1	1.1	1.5
	0.84	1.2	1.0	1.0	1.4
	2.5	1.3	1.2	1.0	1.8
	7.6	1.1	0.76	1.0	0.87
	23	1.2	0.51	1.5	3.2*
	68	0.89	0.26	1.4	4.6*
Hesperetin	0.014	1.0	1.0	1.1	1.6
	0.043	1.4	1.1	1.0	1.4
	0.130	1.1	1.1	1.1	1.6
	0.389	1.2	1.0	1.1	1.3
	1.167	1.0	1.1	1.1	1.5
	3.5	0.79	0.89	0.55	0.94

*Greater than 2-fold activation deemed to be statistically significant

The fold-activation for the agonist assays for each serial dilution of the mixture is shown below in Table 7. The undiluted mixture comprised 1.22 mg/ml of alpha lipoic acid, 0.49 mg/ml of resveratrol, 0.079 mg/ml of curcumin, 0.34 mg/ml of EGCG, 0.37 mg/ml of Olivol®, 0.49 mg/ml of rutin, 7.36 mg/ml of quercetin, and 0.49 mg/ml of hesperetin.

TABLE 7

		PPARα	PPARδ	PPARγ	Nrf2
		Fold-	Fold-	Fold-	Fold-
Test		Activa-	Activa-	Activa-	Activa-
Compound	Fold Dilution	tion	tion	tion	tion

Mixture	243,000	1.2	1.3	0.91	1.0
	81,000	1.1	1.1	1.0	1.4
	27,000	1.3	1.3	1.0	1.4
	9,000	1.2	1.4	1.1	2.0*
	3,000	1.5	1.2	1.2	3.5*
	1,000	1.5	1.2	2.0*	2.7*

*Greater than 2-fold activation deemed to be statistically significant

The percent inhibition and percent live cell for the antagonist assays for each serial dilution of each test compound is shown below in Table 8.

TABLE 8

	Active
	Ingredient
Test	concentration	NF-κB	NF-κB
Compound	in μg/ml	% Inhibition	% Live Cell

Vehicle (DMSO)	0.10%	0.0	0.0
Alpha lipoic	0.47	4.4	100
acid	1.42	3.1	100
	4.25	−0.70	104
	12.7	−6.1	102
	38.23	5.9	104
	114.70	−14	105
Resveratrol	0.031	−9.7	107
	0.09	24	105
	0.28	1.7	100
	0.85	−2.6	100
	2.54	−50	102
	1.81	−28	99
Curcumin	0.007	−10	105
	0.022	−3.4	105
	0.067	−0.29	106
	0.20	22	105
	0.60	30	99
	1.81	51**	97
EGCG	0.033	10	102
	0.098	18	101
	0.30	0.10	105
	0.89	8.4	104
	2.67	−15	106
	8	−29	107
Olivol ®	0.8	27	99
	0.25	−3.8	98
	0.74	−2.2	100
	2.2	13	97
	6.7	13	99
	20	16	98
Rutin	0.12	−3.4	102
	0.37	2.7	102
	1.11	10	99
	3.3	−2.3	98
	10.0	−11	101
	30	−15	97
Quercetin	0.28	−12	103
	0.84	−6	99
	2.5	−12	103
	7.6	18	99
	23	58**	97
	68	85**	77***
Hesperetin	0.014	−6.8	101
	0.043	−11	98
	0.13	−2.5	100
	0.389	−2.1	99
	1.167	14	98
	3.5	39	96

**Greater than 2-fold inhibition deemed to be statistically significant
***Possible cytotoxicity

The fold-activation for the antagonist assays for each serial dilution of the mixture is shown below in Table 9A. The undiluted mixture comprised 1.22 mg/ml of alpha lipoic acid, 0.49 mg/ml of resveratrol, 0.079 mg/ml of curcumin, 0.34 mg/ml of EGCG, 0.37 mg/ml of Olivol®, 0.49 mg/ml of rutin, 7.36 mg/ml of quercetin, and 0.49 mg/ml of Hesperetin.

TABLE 9A

Test		NF-κB	NF-κB
Compound	Fold Dilution	% Inhibition	% Live Cell

Mixture	243,000	20	96
	81,000	11	98
	27,000	11	98
	9,000	−4.7	96
	3,000	−40	97
	1,000	1.9	97

The results for the receptor assays for human PPARα, PPARδ, PPARγ, and Nrf2 in agonist mode, human NF-κB in antagonist mode, and known agonists were analyzed and are presented graphically as FIGS. 5-23. FIG. 5 shows the fold-activation of PPARα for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 6 shows the fold-activation of PPARα for Olivol®, rutin, quercetin, and Hesperetin. FIG. 7 shows the fold-activation of PPARα for the mixture. FIG. 8 shows the fold-activation for a known PPARα agonist, GW590735, as the positive control.
FIG. 9 shows the fold-activation of PPARδ for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 10 shows the fold-activation of PPARδ for Olivol®, rutin, quercetin, and Hesperetin. FIG. 11 shows the fold-activation of PPARδ for the mixture. FIG. 12 shows the fold-activation for a known PPARδ agonist, GW0742, as the positive control.
FIG. 13 shows the fold-activation of PPARγ for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 14 shows the fold-activation of PPARγ for Olivol®, rutin, quercetin, and Hesperetin. FIG. 15 shows the fold-activation of PPARγ for the mixture. The induction concentration of alpha lipoic acid and resveratrol was 114.8 μg/mL and 2.54 μg/mL respectively when they are used alone, but was 1.22 μg/mL and 0.49 μg/mL respectively in the mixture, indicating the synergistic effect of the mixture. FIG. 16 shows the fold-activation for a known PPARγ agonist, rosiglitazone, as the positive control. As shown in the above described figures, a strong activity was observed, particularly for PPARγ compared with PPARα and PPARδ, either by the ingredients alone or the mixture. PPARγ is known to be a potent regulator of lipid and glucose metabolism, and synthetic PPARγ activators such as TZDs were once used as anti-diabetic drugs. Therefore, such findings have clinical relevance in improving metabolic health. FIG. 17 shows the fold-activation of Nrf2 for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 18 shows the fold-activation of Nrf2 for Olivol®, rutin, quercetin, and Hesperetin. FIG. 19 shows the fold-activation of Nrf2 for the mixture. FIG. 20 shows the fold-activation for a known Nrf2 agonist, L-sulphoraphane.
FIG. 21 shows the percent inhibition of human NF-κB in antagonist mode form for alpha lipoic acid, resveratrol, curcumin, and EGCG. FIG. 22 shows the percent inhibition of human NF-κB in antagonist mode form for Olivol®, rutin, quercetin, and Hesperetin. FIG. 23 shows the percent inhibition of human NF-κB in antagonist mode form for the mixture.
An analysis of the data indicated that alpha lipoic acid exhibited very low-level agonist activity against human PPARα, PPARγ, and Nrf2 at the concentrations tested. The data also indicated that resveratrol exhibited very low-level agonist activity against human PPARδ and human Nrf2 and mid-level activity against human PPARγ at the concentrations tested. The data also indicated that curcumin exhibited very low-level antagonist activity against human NF-κB at the concentrations tested. The data also indicated that quercetin exhibited low-level agonist activity against human Nrf2 and very low-level antagonist activity against human NF-κB with some evidence of compounded-induced cytotoxicity at the concentrations tested. Importantly, the data also indicated that the mixture exhibited agonist activity against human PPARγ and human Nrf2 at a concentration much lower than when they were used alone.

Example 2

Phenotypic screening with a specialized strain of C. elegans worm was carried out using two test formulations of the disclosed nutritional supplement compositions to assess their effect on epigenetic anti-ageing activity. A first test formulation comprised the composition as described below in Table 9B and was labeled as “N356.” A second test formulation comprised a combination of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and Hesperetin as described below in Table 9C and was labeled as “N357.” Each test formulation was tested over a range of concentrations. Dilutions of the test formulations were applied to individual populations of C. elegans and the lifespan of each population was monitored. Any changes in the lifespan of an individual population compared to a control population were recorded and correlated to the respective test formulation (N356 or N357) and the respective dilution (0.1 mg/ml, 1 mg/ml, and 10 mg/ml).

TABLE 9B

Active Ingredient:	mg:

Mixed Carotenoids (alpha, beta, gamma carotene and	0.10
lycopene)
Beta carotene (2150 IU tab)	1.29
Retinyl Acetate (750 IU)	0.26
Vitamin C (Poly C, Ca, K, Mg and Zn Ascorbates)	100.00
Vitamin D3 (Cholecalciferol) [500 IU/tab]	0.0125
Vitamin E (d-alpha-tocopheryl succ. 50 IU)	41.30
Mixed Tocopherols	20.00
Vitamin K1	0.12
Vitamin K2 (menaquinone, MK-7)	0.02
Vitamin B1 (thiamin HCL)	7.50
Vitamin B2 (riboflavin)	7.50
Niacin	2.50
Niacinamide	7.50
Vitamin B6 (pyridoxine HCL)	8.00
Folic Acid	0.15
Vitamin B12 (cyanocobalamin)	0.05
Biotin	0.08
Pantothenic Acid	22.50
Alpha Lipoic Acid	25.00
Resveratrol	10.00
Curcumin Phytosome Complex (containing 3.25 mg	18.06
curcuminoids)
Green Tea Extract (standardized to EGCG)	17.50
Olivol ® (Olive Fruit Extract)	7.50
Rutin	10.00
Quercetin Dihydrate	15.00
Hesperetin	10.00
Inositol	32.00
Choline bitartrate	62.50
Coenzyme Q-10	3.00
Lutein	0.15
Lycopene	0.25

TABLE 9C

Active Ingredient:	mg:

Alpha Lipoic Acid	25.00
Resveratrol	10.00
Curcumin Phytosome Complex (containing 3.25 mg curcuminoids)	18.06
Green Tea Extract (standardized to EGCG)	17.50
Olivol (Olive Fruit Extract)	7.50
Rutin	10.00
Quercetin Dihydrate	15.00
Hesperidin	10.00

The N356 and N357 formulations were each prepared as individual 100 mg/ml stock solutions in dimethyl sulfoxide (DMSO). Serial dilutions of each stock solution were then prepared at 0.1 mg/ml, 1 mg/ml, and 10 mg/ml in DMSO. Control solutions of DMSO only were also prepared. Synchronous aged adult populations of C. elegans strain CB5586 worms were prepared. The CB5586 strain comprises a pharyngeal GFP (green fluorescent protein) tag and a mutation in the bus-5 gene. The pharyngeal GFP tag allows for fluorescent images of the worm populations to be taken. The mutation in the bus-5 gene causes the loss of normal cuticle antigens that permits the cuticle of the worms to become permeable to the test formulations and allow direct uptake of the test formulations to avoid interaction of the test formulations with protective mechanisms of the gut channel. Each serial dilution of each test formulation was added to a separate population of the prepared worms. The control solutions were also added to separate populations of the prepared worms. The populations of the prepared worms were then maintained on standard nematode growth media (NGM) agar plates at 20° C. with sufficient food (Escherichia coli strain OP-50).
Each worm population was then monitored by fluorescent imaging to determine the number of living and dead worms as a function of time. The fluorescent imaging was analyzed by software that recognized and counted worms based on their fluorescent intensity compared to background fluorescence. Living worms were automatically distinguished by the software from dead worms based on the degree of movement they exhibited between consecutive fluorescent images. The ability to distinguish between living and dead worms allowed the number of living worms and cumulative number of dead worms to be monitored against time. Worm populations from a selection of fluorescent images were manually checked to verify that the software had accurately counted the number of living and dead worms.
The counts of the living and dead worms as a function of time were then analyzed for each of the worm populations and used to prepare Kaplan-Meier survival curves and associated statistics for each of the worm populations. FIG. 24 shows an ideal Kaplan-Meier survival curve for a control population and a population exposed to an ideal test compound, “Compound A.” The health span extension is shown as the length of time after the last mitotic division that 95% of the test population remains viable when compared to the control population. The median lifespan is shown as the length of time after the last mitotic division that 50% of the test population remains viable when compared to the control population. The maximum lifespan extension is shown as the length of time after the last mitotic division that 5% of the test population remains viable when compared to the control population.
FIG. 25 show a Kaplan-Meier survival curve for the worm population tested with N356 at the 0.1 mg/ml concentration. FIG. 26 show a Kaplan-Meier survival curve for the worm population tested with N356 at the 1.0 mg/ml concentration. FIG. 27 show a Kaplan-Meier survival curve for the worm population tested with N356 at the 10 mg/ml concentration. FIG. 28 shows a dose-dependent extension of lifespan for N356 at 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measured as a function of age at 20% mortality.
FIG. 29 show a Kaplan-Meier survival curve for the worm population tested with N357 at the 0.1 mg/ml concentration. FIG. 30 show a Kaplan-Meier survival curve for the worm population tested with N357 at the 1.0 mg/ml concentration. FIG. 31 show a Kaplan-Meier survival curve for the worm population tested with N357 at the 10 mg/ml concentration. FIG. 32 shows a dose-dependent extension of lifespan for N357 at 0.1 mg/ml, 1.0 mg/ml, and 10 mg/ml compared to DMSO for health span measured as a function of age at 20% mortality.
The counts of the living and dead worms as a function of time and the Kaplan-Meier survival curves and the associated statistics were then analyzed for each of the worm populations. The analysis included determining mean and median lifespan. The analysis included a non-parametric test, the Log-Rank test, which compares two survival functions for the overall lifespan assay and provides a reliable p-value summarizing the whole experiment. The analysis also included Fisher's Exact Test that calculated the significance of survival function comparisons at multiple specific time points throughout the experiment, rather than for the overall lifespan. The results of the analysis are shown below in Tables 10-17. Table 10 shows the restricted mean lifespan for the N356 treated worm population compared to the DMSO control. Table 11 shows the restricted mean lifespan for the N357 treated worm population compared to the DMSO control. Table 12 shows the population age in days at given percent mortalities for the N356 treated worm population compared to the DMSO control. Table 13 shows the population age in days at given percent mortalities for the N357 treated worm population compared to the DMSO control. Table 14 shows the Log-Rank Test results for the N356 treated worm population compared to the DMSO control. Table 15 shows the Log-Rank Test results for the N356 treated worm population compared to the DMSO control. Table 16 shows the Fisher's Exact Test results for the N356 treated worm population compared to the DMSO control. Table 17 shows the Fisher's Exact Test results for the N357 treated worm population compared to the DMSO control.

TABLE 10

			95% Confidence
Test Formulation	# of Days	Standard Error	Interval

DMSO	13.23	0.08	13.06~13.39
N356 at 0.1 mg/ml	15.18	0.14	14.90~15.45
N356 at 1.0 mg/ml	10.56	0.36	9.85~11.27
N356 at 10 mg/ml	10.87	0.24	10.40~11.34

TABLE 11

			95% Confidence
Test Formulation	# of Days	Standard Error	Interval

DMSO	13.23	0.08	13.06~13.39
N357 at 0.1 mg/ml	14.40	0.15	14.11~14.70
N357 at 1.0 mg/ml	9.81	0.41	9.00~10.61
N357 at 10 mg/ml	11.26	0.26	10.77~11.76

TABLE 12

Test Formulation	25%	50%	75%	90%	100%

DMSO	11	14	16	—	—
N356 at 0.1 mg/ml	14	16	—	—	—
N356 at 1.0 mg/ml	7	11	14	18	—
N356 at 10 mg/ml	7	11	16	—	—

TABLE 13

Test Formulation	25%	50%	75%	90%	100%

DMSO	11	14	16	—	—
N357 at 0.1 mg/ml	11	16	18	—	—
N357 at 1.0 mg/ml	9	11	14	16	18
N357 at 10 mg/ml	9	11	14	18	—

TABLE 14

Condition	Chi²	P-value	Bonferroni P-value

DMSO vs. N356 at 0.1 mg/ml	105.81	0.0e+00	0.0e+00
DMSO vs. N356 at 1.0 mg/ml	47.47	0.0e+00	0.0e+00
DMSO vs. N356 at 10 mg/ml	51.94	0.0e+00	0.0e+00

TABLE 15

Condition	Chi²	P-value	Bonferroni P-value

DMSO vs. N357 at 0.1 mg/ml	45.16	0.0e+00	0.0e+00
DMSO vs. N357 at 1.0 mg/ml	67.03	0.0e+00	0.0e+00
DMSO vs. N357 at 10 mg/ml	45.17	0.0e+00	0.0e+00

TABLE 16

	P-value	P-value	P-value	P-value
Condition	at 25%	at 50%	at 75%	at 90%

DMSO vs. N356 at 0.1 mg/ml	2.7e−12	2.5e−12	1.9e−12	1.5e−12
DMSO vs. N356 at 1.0 mg/ml	5.0e−08	5.0e−08	3.3e−06	0.0093
DMSO vs. N356 at 10 mg/ml	2.0e−12	3.5e−11	0.0001	0.0591

TABLE 17

	P-value	P-value	P-value	P-value
Condition	at 25%	at 50%	at 75%	at 90%

DMSO vs. N357 at 0.1 mg/ml	6.6e−11	3.0e−11	2.2e−07	9.7e−07
DMSO vs. N357 at 1.0 mg/ml	1.3e−11	1.3e−11	5.2e−08	0.0001
DMSO vs. N357 at 10 mg/ml	9.8e−11	9.8e−11	1.2e−06	0.0012

The analysis of the phenotypic screen indicated that both N356 and N357 displayed anti-ageing activity. In particular, at the 0.1 mg/ml concentration, the worm population treated with N356 and the worm population treated with N357 both showed statistically significant increases in chronological lifespan. Treatment with N356 resulted in a statistically significant improvement in mean life span of about 9%, a maximum improvement in lifespan of about 14.3% at 50% mortality, and a maximum improvement in survival up to about 25% between days 11 and 14 (e.g., the mid-lifespan and the late-life span). Likewise, treatment with N357 resulted in a statistically significant improvement in mean lifespan of about 9%, a maximum improvement in lifespan of about 14.3% at 50% mortality, and a maximum improvement in survival up to about 16% between days 11 and 14 (mid-lifespan and late-lifespan). The maximum effect was seen in the combination formulation N356. Both treatments also demonstrated significant improvements to lifespan during the early stages of the population survival curve that lay between health span and median lifespan.
The analysis of the N356 populations indicated that of the 0.1 mg/ml, the 1.0 mg/ml, and the 10 mg/ml concentrations that the 0.1 mg/ml concentration appeared to be the optimum dose. The N356 0.1 mg/ml concentration treatment resulted in a statistically significant improvement in mean life span of 15%, a maximum improvement in lifespan of 27% at 25% mortality, and a maximum improvement in survival up to 25% between days 11 and 14. The Log-Rank test results and the Fisher's Exact test results showed significance overall for the length of the study and for each individual time point within the study for the N356 0.1 mg/ml concentration. The analysis of the N357 populations indicated that of the 0.1 mg/ml, the 1.0 mg/ml, and the 10 mg/ml concentrations that the 0.1 mg/ml concentration appeared to be the optimum dose. The N357 0.1 mg/ml concentration treatment resulted in a statistically significant improvement in mean life span of 9%, a maximum improvement in lifespan of 14.3% at 50% mortality, and a maximum improvement in survival up to 16% between days 11 and 14. The Log-Ran test results and the Fisher's Exact test results showed significance overall for the length of the study and for each individual time point within the study for the N357 0.1 mg/ml concentration.
Both the N356 and the N357 treatments showed a dose dependent effect as seen in FIGS. 28 and 32. The threshold for a positive effect on lifespan seemed to lie somewhere above 1.0 mg/ml and thus 0.1 mg/ml was accepted as the optimum dose of those concentrations that were tested. For both N356 and N357, when the concentration was increased to 1.0 mg/ml, the treatments caused a decrease in lifespan compared to a control. Similarly, at 10 mg/ml, a decrease in lifespan of the respective worm populations was seen for both N356 and N357. It is possible that treatments with concentrations of N356 and N357 below 0.1 mg/ml may also increase lifespan. This data indicate that the components of the formulation corresponding to antioxidants also increased lifespan above the effect produced by the herbal components of the formulation. The data indicate that there is a synergistic effect on lifespan from the administration of the antioxidant components and the herbal components. In some cases, there can be a complimentary effect on lifespan from the administration of the antioxidant components and the herbal components.

Example 3

Phenotypic screening was carried out as described in EXAMPLE 2 for various individual compounds of the N357 formulation. Individual compounds were assayed to determine any possible individual contribution that an individual compound may have to overall anti-ageing activity. Solutions of resveratrol, alpha lipoic acid, hesperidin (hesperetin), quercetin, and rutin hydrate were prepared at concentrations of 0.1 mg/ml and 10 mg/ml in DMSO. Resveratrol was sourced from Sigma Aldrich at ≧99% HPLC purity and was assigned a sample number of N108.
Alpha lipoic acid was sourced from PureBulk™ USA as a racemic mix of R and S stereoisomers and was assigned a sample number of N198. Hesperidin was sourced from Sigma Aldrich at ≧80% purity and was assigned a sample number of N347. Quercetin was sourced from Tocris Bioscience at ≧98% HPLC purity and was assigned a sample number of N104. Rutin hydrate was sourced from Sigma Aldrich at ≧94% HPLC purity and was assigned a sample number of N346. The counts of the living and dead worms as a function of time were analyzed for each of the worm populations and used to prepare Kaplan-Meier survival curves and associated statistics for each of the worm populations as described above. Log-Rank test and Fisher's Exact test were also performed.
FIGS. 33 to 37 show Kaplan-Meier survival curves for the worm populations treated with resveratrol, alpha lipoic acid, hesperidin, quercetin, and rutin hydrate. FIG. 33 show a Kaplan-Meier survival curve for the worm population tested with N108 (resveratrol) at the 0.1 mg/ml and 10 mg/ml concentrations. FIG. 34 show a Kaplan-Meier survival curve for the worm population tested with N198 (alpha lipoic acid) at the 0.1 mg/ml and 10 mg/ml concentrations. FIG. 35 show a Kaplan-Meier survival curve for the worm population tested with N347 (hesperidin) at the 0.1 mg/ml and 10 mg/ml concentrations. FIG. 36 show a Kaplan-Meier survival curve for the worm population tested with N104 (quercetin) at the 0.1 mg/ml and 10 mg/ml concentrations. FIG. 37 show a Kaplan-Meier survival curve for the worm population tested with N346 (rutin hydrate) at the 0.1 mg/ml and 10 mg/ml concentrations.
The mean and median lifespan for each worm population treated with resveratrol, alpha lipoic acid, hesperidin, quercetin, and rutin hydrate are listed below in Table 18. The age in days at 25%, 50%, 75%, 90%, and 100% mortality are listed below in Table 19. The Log-Rank test results for each worm population compared to the DMSO control are shown below in Table 20. The Fisher's Exact Test results for each worm population compared to the DMSO control are shown below in Table 21.

TABLE 18

Name	Days	Std. error	95% C.I.

DMSO	13.23	0.08	13.06~13.39
N108 10 mg/ml (resveratrol)	15.16	0.15	14.87~15.45
N108 0.1 mg/ml (resveratrol)	13.57	0.19	13.20~13.95
N198 10 mg/ml (alpha lipoic acid)	14.85	0.14	14.57~15.13
N198 0.1 mg/ml (alpha lipoic acid)	14.89	0.18	14.55~15.24
N347 10 mg/ml (hesperidin)	14.19	0.16	13.87~14.51
N347 0.1 mg/ml (hesperidin)	14.55	0.16	14.23~14.86
N104 10 mg/ml (quercetin)	12.54	0.27	12.02~13.07
N104 0.1 mg/ml (quercetin)	12.17	0.20	11.78~12.57
N346 10 mg/ml (rutin hydrate)	13.43	0.22	13.01~13.86
N346 0.1 mg/ml (rutin hydrate)	12.03	0.21	11.61~12.45

TABLE 19

	25%	50%	75%	90%	100%
	mor-	mor-	mor-	mor-	mor-
Name	tality	tality	tality	tality	tality

DMSO	11	14	16	—	—
N108 10 mg/ml (resveratrol)	14	16	18	—	—
N108 0.1 mg/ml (resveratrol)	11	14	18	—	—
N198 10 mg/ml (alpha lipoic acid)	14	16	18	—	—
N198 0.1 mg/ml (alpha lipoic acid)	14	16	18	—	—
N347 10 mg/ml (hesperidin)	11	14	18	—	—
N347 0.1 mg/ml (hesperidin)	11	16	18	—	—
N104 10 mg/ml (quercetin)	9	14	16	—	—
N104 0.1 mg/ml (quercetin)	9	11	14	18	—
N346 10 mg/ml (rutin hydrate)	11	14	16	—	—
N346 0.1 mg/ml (rutin hydrate)	9	11	14	18	—

TABLE 20

Condition	Chi²	P-value	Bonferroni P-value

DMSO vs. N108 at 10 mg/ml	86.88	0.0e+00	0.0e+00
DMSO vs. N108 at 0.1 mg/ml	4.59	0.0321	0.0642
DMSO vs. N198 at 10 mg/ml	60.49	0.0e+00	0.0e+00
DMSO vs. N198 at 0.1 mg/ml	55.75	0.0e+00	0.0e+00
DMSO vs. N347 at 10 mg/ml	19.37	1.1e−05	2.2e−05
DMSO vs. N347 at 0.1 mg/ml	45.04	0.0e+00	0.0e+00
DMSO vs. N104 at 10 mg/ml	4.98	0.0256	0.0512
DMSO vs. N104 at 0.1 mg/ml	26.70	2.4e−07	4.7e−07
DMSO vs. N346 at 10 mg/ml	0.23	0.6322	1.0000
DMSO vs. N346 at 0.1 mg/ml	28.00	1.2e−07	2.4e−07

TABLE 21

	P-value	P-value	P-value	P-value
Condition	at 25%	at 50%	at 75%	at 90%

DMSO vs. N108 at 10 mg/ml	3.1e−12	2.7e−12	2.9e−12	5.6e−09
DMSO vs. N108 at 0.1 mg/ml	0.0517	0.0108	0.1303	0.1226
DMSO vs. N198 at 10 mg/ml	3.2e−12	3.2e−12	9.1e−08	1.1e−05
DMSO vs. N198 at 0.1 mg/ml	2.7e−12	2.6e−12	2.6e−12	4.4e−06
DMSO vs. N347 at 10 mg/ml	2.6e−08	7.4e−08	0.0028	0.0313
DMSO vs. N347 at 0.1 mg/ml	1.1e−09	2.5e−12	2.1e−07	8.5e−06
DMSO vs. N104 at 10 mg/ml	0.0330	0.5557	0.0754	0.2111
DMSO vs. N104 at 0.1 mg/ml	9e−08	9.8e−08	0.0002	0.0031
DMSO vs. N346 at 10 mg/ml	0.8993	0.1912	0.7094	1.0000
DMSO vs. N346 at 0.1 mg/ml	2.7e−07	2.7e−07	2.7e−06	0.0031

The analysis indicated that resveratrol (N108), alpha lipoic acid (N198), and hesperidin (N347) all showed significant positive and dose dependent increase of chronological lifespan. Quercetin (N104) and rutin (N346) appeared to show significant negative effect on chronological lifespan at certain tested concentrations. FIG. 33 shows that resveratrol appeared to extend lifespan by about 15% at 10 mg/ml. FIG. 34 shows that alpha lipoic acid appeared to extend lifespan by about 12.5% at both 0.1 mg/ml and 10 mg/ml. FIG. 35 shows that hesperidin appeared to extend lifespan by about 10% at 0.1 mg/ml. FIG. 36 shows that quercetin, at least at the concentrations tested and under the conditions tested, appeared to decrease lifespan by about 9% at 0.1 mg/ml. FIG. 37 shows that rutin hydrate, at least at the concentrations tested and under the conditions tested, appeared to decrease lifespan by about 9% at 0.1 mg/ml. The summary of results for the worm populations treated with resveratrol, alpha lipoic acid, hesperidin, quercetin, and rutin hydrate compared to DMSO control are summarized below in Table 22.

TABLE 22

		% Change in
	Statistically Significant	Mean
Compound	Result	Lifespan

0.1 mg/ml (resveratrol)	No statistical difference	N/A
10 mg/ml (resveratrol)	Statistically significant	+15%
	increase
0.1 mg/ml (alpha lipoic acid)	Statistically significant	+12.5%
	increase

10 mg/ml (alpha lipoic acid)	Statistically significant	+12.5%
	increase
0.1 mg/ml (hesperidin)	Statistically significant	+10%
	increase

10 mg/ml (hesperidin)	No statistical difference	N/A
0.1 mg/ml (quercetin)	Statistically significant	−9%
	decrease

10 mg/ml (quercetin)	No statistical difference	N/A
0.1 mg/ml (rutin hydrate)	Statistically significant	−8%
	decrease

10 mg/ml (rutin hydrate)	No statistical difference	N/A

N/A = Not applicable

Example 4

In vitro assays of Nrf2 signaling activity were carried out for each of alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin. The assays were carried out with a fluorescent reporter assay that utilized a human retinal epithelial cell line. The assays also included validation by RT-PCR (reverse transcription polymerase chain reaction) of Nrf2 target genes. The human retinal epithelial cell line was purchased from ATCC (American Type Cell Culture, Manassas, Va.) and was configured to function as a fluorescent reporter assay. Test solutions of each of the alpha lipoic acid, resveratrol, curcumin, EGCG, Olivol®, rutin, quercetin, and hesperetin were prepared by serial dilution. Control solutions were also prepared. Positive control solutions of a known Nrf2 agonist, L-sulphoraphane was also prepared. Each test solution was then assayed for Nrf2 signaling activity using the fluorescent reporter assay and compared to control assays. The positive control solutions were also tested.
FIG. 38 shows the fold-activation of Nrf2 by each test solution compared to the control. The minimum inducing concentrations of alpha lipoic acid was 1.75 of resveratrol was 1.0 and of quercetin was 0.55 FIG. 39 shows the fold-activation of Nrf2 by alpha lipoic acid at 100 μM and 30 μM against a control. The fold activation of 5 μM sulforaphane, as a positive control is also shown. Alpha lipoic acid demonstrated activation of Nrf2 at both 100 μM and 30 FIG. 40 shows the fold-activation of Nrf2 by quercetin at 10 μM and 1 μM against a control. The fold activation of 5 μM sulforaphane, as a positive control is also shown. Quercetin demonstrated activation of Nrf2 at both 10 μM and 1 with more activation at the 1 μM concentration. FIG. 41 shows the fold-activation of Nrf2 by resveratrol at 10 μM and 1 μM against a control. The fold activation of 5 μM sulforaphane, as a positive control is also shown. Resveratrol demonstrated activation of Nrf2 at both 10 μM and 1 with more activation at the 10 μM concentration.
The terms “a,” “an,” “the” and similar referents used in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.
It is contemplated that numerical values, as well as other values that are recited herein are modified by the term “about”, whether expressly stated or inherently derived by the discussion of the present disclosure. As used herein, the term “about” defines the numerical boundaries of the modified values so as to include, but not be limited to, tolerances and values up to, and including the numerical value so modified. That is, numerical values can include the actual value that is expressly stated, as well as other values that are, or can be, the decimal, fractional, or other multiple of the actual value indicated, and/or described in the disclosure.
Groupings of alternative elements or embodiments of the disclosure disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
Certain embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.
In closing, it is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

Claims

We claim:

1. A nutritional supplement for reducing free radical damage, the supplement comprising:

an upregulating compound mixture configured to upregulate an endogenous antioxidant system, comprising at least one of;

an exogenous antioxidant mixture; and

a mineral mixture.

2. The supplement of claim 1, wherein the upregulating compound mixture comprises one or more of alpha lipoic acid, resveratrol, curcumin, and EGCG.

3. The supplement of claim 1, wherein the upregulating compound mixture comprises one or more of Olivol®, rutin, quercetin, and hesperetin.

4. The supplement of claim 1, wherein the exogenous antioxidant mixture comprises one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.

5. A nutritional supplement for reducing free radical damage, the supplement comprising:

a first vehicle comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture; and

a second vehicle comprising a mineral mixture.

6. The supplement of claim 5, wherein the first vehicle is a single solid tablet and wherein the second vehicle is a single solid tablet.

7. The supplement of claim 5, wherein the upregulating compound mixture comprises one or more of alpha lipoic acid, resveratrol, curcumin, and EGCG.

8. The supplement of claim 5, wherein the upregulating compound mixture comprises one or more of Olivol®, rutin, quercetin, and hesperetin.

9. The supplement of claim 5, wherein the exogenous antioxidant mixture comprises one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.

10. A method for manufacturing a nutritional supplement for reducing free radical damage, the method comprising:

combining a first vehicle comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture, with a second vehicle comprising a mineral mixture,

wherein the upregulating compound mixture is configured to upregulate an endogenous antioxidant system to reduce free radical damage.

11. The method of claim 10, wherein the upregulating compound mixture comprises one or more of alpha lipoic acid, resveratrol, curcumin, and EGCG.

12. The method of claim 10, wherein the upregulating compound mixture comprises one or more of Olivol®, rutin, quercetin, and hesperetin.

13. The method of claim 10, wherein the exogenous antioxidant mixture comprises one or more of mixed carotenoids, beta carotene, retinyl acetate, vitamin C, vitamin D3, vitamin E, mixed tocopherols, vitamin K1, vitamin K2, vitamin B1, vitamin B2, niacin, niacinamide, vitamin B6, folic acid, vitamin B12, biotin, pantothenic acid, inositol, choline bitartrate, coenzyme Q-10, lutein, and lycopene.

14. The method of claim 10, wherein the mineral mixture comprises one or more of calcium citrate, calcium ascorbate, magnesium citrate, magnesium ascorbate, potassium iodine, zinc citrate, L-selenomethionine, sodium selenite, copper gluconate, manganese gluconate, chromium polynicotinate, molybdenum citrate, boron citrate, calcium silicate, vanadium citrate, ultra-trace minerals, and N-acetyl-L-cysteine

15. The method of claim 10, wherein the endogenous antioxidant system comprises a transcription factor.

16. The method of claim 15, wherein the transcription factor comprises one or more of Nrf2, NF-κB, PPARα, PPARβ/δ, and PPARγ.

17. The method of claim 16, wherein the transcription factor promotes transcription of an antioxidant gene.

18. The method of claim 17, wherein the antioxidant gene comprises a Phase II gene.

19. The method of claim 17, wherein the antioxidant gene comprise one of more of a NQO1 gene, a GCL gene, a sulfiredoxin 1 (SRXN1) gene, a thioredoxin reductase 1 (TXNRD1) gene, a HO-1 gene, a GST family gene, and an UDP-glucuronosyltransferase (UGT) family gene.

20. A kit comprising:

a nutritional supplement comprising a first vehicle comprising an upregulating compound mixture configured to upregulate an endogenous antioxidant system and an exogenous antioxidant mixture and a second vehicle comprising a mineral mixture; and

a container.