WO2017200911A1 - Compositions for activating mitochondria - Google Patents

Abstract

Compositions including Rhodiola rosea, coenzyme Q₁₀, grape extract, alphalipoic acid, and/or quercetin are provided. The compositions may be used for activating mitochondria in a subject. The compositions may modulate the expression of at least one of NRF1, cytochrome C, and/or TFAM in a manner that is independent of PGC-1α. Methods of using the compositions are also provided.

Description

COMPOSITIONS FOR ACTIVATING MITOCHONDRIA

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/337,238, filed on May 16, 2016, the entire contents of which are fully incorporated herein by reference.

TECHNICAL FIELD

[0002] The present disclosure relates to compositions for use in activating mitochondria. More particularly, the disclosure relates to compositions including Rhodiola rosea, coenzyme Qio, grape extract, alpha-lipoic acid, and/or quercetin. Methods of using the compositions are also disclosed.

BACKGROUND

[0003] Mitochondria are cellular organelles, which can produce energy (e.g., in the form of ATP). During the production of energy, mitochondria may release free radicals.

Mitochondria can also be involved in cell maintenance and cell death. Through fission and/or fusion, mitochondria can constantly and/or dynamically change. Exercise, exposure to cold temperature, and caloric restriction can induce the production of peroxisome proliferator- activated receptor gamma coactivator 1 -alpha (PGC-la), a transcriptional co-activator, which in turn can upregulate the production of nuclear respiratory factor 1 (NRF1), a transcription factor. NRF1 can play a regulatory role in mitochondrial biogenesis and replacement in response to energy deficit.

[0004] NRFl can also activate a series of genes, including the Von Hippel-Lindau tumor suppressor (pVHL), cytochrome c, and mitochondrial transcription factor A (TFAM), which can ultimately effect the replication of mitochondrial DNA and the assembly of new mitochondria. The ability of NRFl to induce gene expression for mitochondria biogenesis can be dependent on the presence of PGC-la.

BRIEF DESCRIPTION OF THE DRAWINGS [0005] The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.

[0006] FIG. 1 is a graph showing the effects of two candidate compounds and the NRFl blend (NRF-1 Combo) on cell viability at various doses in HEK 293 cells treated for 24 hours.

[0007] FIG. 2A is a graph showing the effect of five candidate compounds and the NRFl blend on expression of MTCOl (cytochrome c), NRFl, PGC-la, and TFAM in HEK 293 following 6 hours of exposure. Data are presented as fold expression compared to untreated controls (expression = 1.0).

[0008] FIG. 2B is a graph showing the effect of the five candidate compounds and the NRFl blend on expression of MTCOl, NRFl, PGC-la, and TFAM in HEK 293 following 24 hours of exposure. Data are presented as fold expression compared to untreated controls (expression = 1.0).

[0009] FIG. 3A is a graph showing the effect of the five candidate compounds and the NRFl blend (at the doses indicated in parentheses) on expression of MTCOl, NRFl, PGC- la, and TFAM in HEK 293 following 6 hours of exposure.

[0010] FIG. 3B is a graph showing the effect of the five candidate compounds and the NRFl blend (at the doses indicated in parentheses) on expression of MTCOl, NRFl, PGC- la, and TFAM in HEK 293 following 24 hours of exposure.

[0011] FIG. 4 is a diagram depicting a process of pooling the RNA extracted from each treatment group into three subgroups (three mice per treatment group) and taking each subgroup forward into RNA-Seq as its own sample.

[0012] FIG. 5A is a graph showing the effect of the five candidate compounds (at the doses indicated in brackets) on expression of MTCOl, NRFl, PGC-la, and TFAM in HEK 293 following 6 hours of exposure.

[0013] FIG. 5B is a graph showing the effect of the five candidate compounds (at the doses indicated in brackets) on expression of MTCOl, NRFl, PGC-la, and TFAM in HEK 293 following 24 hours of exposure. [0014] FIG. 6A is a graph showing the effect of Rhodiola rosea on cell viability at various doses in HEK 293 cells treated for 24 hours.

[0015] FIG. 6B is a graph showing the effects of alpha-lipoic acid (blue) and quercetin (grey) on cell viability at various doses in HEK 293 cells treated for 24 hours.

DETAILED DESCRIPTION

[0016] It will be readily understood that the embodiments, as generally described herein, are exemplary. The following more detailed description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps or actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified.

[0017] Amounts, concentrations, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also all the individual numerical values or sub-ranges encompassed within that range, as if each numerical value and sub-range were explicitly recited. For example, an amount of from 1 mg to 200 mg should be interpreted to include not only the explicitly recited limits of 1 mg and 200 mg, but also individual amounts such as 2 mg, 3 mg, 4 mg, and sub-ranges such as 10 mg to 50 mg, 20 mg to 100 mg, etc. Unless otherwise stated, all ranges include both endpoints.

[0018] As used herein, a "subject" refers to a mammal, such as a human, but can also be an animal, for example, a domestic animal (e.g., a dog, a cat, etc.), a farm animal (e.g. , a cow, a sheep, a pig, a horse, etc.) and a laboratory animal (e.g., a rat, a mouse, a guinea pig, etc.).

[0019] A first aspect of the disclosure relates to a composition. The composition may include at least one of Rhodiola rosea extract, coenzyme Q₁₀, grape extract, alpha-lipoic acid, and/or quercetin. In some embodiments, the composition may include Rhodiola rosea extract, coenzyme Q_lo, grape extract, alpha-lipoic acid, and quercetin. [0020] The composition may be configured as a single daily dose for a human subject. In certain embodiments, the single daily dose of the composition may include about 220 mg of Rhodiola rosea extract. In certain other embodiments, the single daily dose of the composition may include from about 25 mg to about 500 mg of Rhodiola rosea extract, from about 75 mg to about 425 mg of Rhodiola rosea extract, from about 125 mg to about 350 mg of Rhodiola rosea extract, from about 175 mg to about 300 mg of Rhodiola rosea extract, from about 200 mg to about 250 mg of Rhodiola rosea extract, or another suitable amount of Rhodiola rosea extract.

[0021] The Rhodiola rosea extract may include at least one of rosavins (a cinnamyl alcohol glycoside) and/or salidroside (a glucoside of tyrosol). In various embodiments, the Rhodiola rosea extract may include about 3% rosavins weight by weight of dry extract. In various other embodiments, the Rhodiola rosea extract may include from about 0.1% to about 10% rosavins, from about 0.5% to about 7.5% rosavins, from about 1% to about 5% rosavins, from about 2% to about 3% rosavins, or another suitable percentage of rosavins. In some embodiments, the Rhodiola rosea extract may include about 1% salidroside weight by weight of dry extract. In some other embodiments, the Rhodiola rosea extract may include from about 0.05% to about 10% salidroside, from about 0.1% to about 7.5% salidroside, from about 0.5% to about 5% salidroside, from about 0.75% to about 2.5% salidroside, from about 0.9% to about 1.5% salidroside, or another suitable percentage of salidroside.

[0022] In certain embodiments, the single daily dose of the composition may include about 100 mg of coenzyme Qio. In certain other embodiments, the single daily dose of the composition may include from about 10 mg to about 400 mg of coenzyme Qio, from about 25 mg to about 300 mg of coenzyme Qio, from about 50 mg to about 200 mg of coenzyme Qio, from about 75 mg to about 150 mg of coenzyme Qio, or another suitable amount of coenzyme

[0023] The grape extract may be obtained from at least one of a seed, a pulp, and/or a skin of the grape. In some embodiments, the grape extract may be obtained from the seed, the pulp, and the skin of the grape. In various embodiments, the single daily dose of the composition may include about 70 mg of grape extract. In certain other embodiments, the single daily dose of the composition may include from about 10 mg to about 300 mg of grape extract, from about 20 mg to about 200 mg of grape extract, from about 40 mg to about 150 mg of grape extract, from about 60 mg to about 100 mg of grape extract, or another suitable amount of grape extract.

[0024] In various embodiments, the alpha-lipoic acid may be synthetic alpha-lipoic acid. In various other embodiments, the alpha-lipoic acid may not be synthetic alpha-lipoic acid. In some embodiments, the single daily dose of the composition may include about 45 mg of alpha-lipoic acid. In some other embodiments, the single daily dose of the composition may include from about 5 mg to about 300 mg of alpha-lipoic acid, from about 15 mg to about 200 mg of alpha-lipoic acid, from about 25 mg to about 100 mg of alpha-lipoic acid, from about 35 mg to about 60 mg of alpha-lipoic acid, or another suitable amount of alpha-lipoic acid.

[0025] In certain embodiments, the quercetin may include quercetin (dihydrate). In various embodiments, the single daily dose of the composition may include about 20 mg of quercetin or quercetin (dihydrate). In various other embodiments, the single daily dose of the composition may include from about 1 mg to about 200 mg of quercetin or quercetin (dihydrate), from about 5 mg to about 100 mg of quercetin or quercetin (dihydrate), from about 10 mg to about 50 mg of quercetin or quercetin (dihydrate), from about 15 mg to about 30 mg of quercetin or quercetin (dihydrate), or another suitable amount of quercetin or quercetin (dihydrate).

[0026] In some embodiments, the composition may be formulated as one or more of a capsule, a tablet, or a powder. In some other embodiments, the composition may be formulated as a liquid drink. In yet other embodiments, the composition may be packaged for single use.

[0027] Another aspect of the disclosure relates to a method of using the composition to modulate the expression of at least one of NRF1 , cytochrome C, and/or TFAM. In some embodiments, the composition may modulate the expression of at least one of NRF1, cytochrome C, and/or TFAM. For example, the composition may enhance or improve the expression of at least one of NRF 1, cytochrome C, and/or TFAM.

[0028] In certain embodiments, the composition may modulate mitochondrial biogenesis and/or maintenance. For example, the composition may enhance or improve mitochondrial biogenesis and/or maintenance. The composition may modulate mitochondrial biogenesis and/or maintenance via NRFl, cytochrome C, and/or TFAM. In various embodiments, the composition may modulate the expression of at least one of NRF1, cytochrome C, and/or TFAM in a manner, or via a pathway, that is independent of or substantially independent of PGC-Ια and/or the activation of PGC-la.

[0029] In some embodiments, the composition may activate the expression of NRF1 at or to a level that is greater than a sum of the levels of NRF1 activation that may be obtained individually by each component (e.g., Rhodiola rosea extract, coenzyme Q₁₀, grape extract, alpha-lipoic acid, and quercetin) of the composition. For example, the composition may have a synergistic effect on the activation of NRF1 expression. Stated another way, the effect of the composition on the activation of NRF1 expression may not merely be an additive effect of the individual components of the composition.

[0030] In certain embodiments, the composition may increase the amount of ATP in the cells of a subject. Increased amounts of ATP in the cells of the subject may be an indication of improved mitochondria function. In certain embodiments, the composition may increase mitochondria protein synthesis in the hearts of old rodents (e.g., rodents that are at least about 9 months of age).

[0031] In some embodiments, the composition may be configured to inhibit or slow aging in the subject. In various embodiments, the composition may be configured to increase stamina in the subject. The certain embodiments, the composition may be configured to increase libido in the subject.

EXAMPLES

[0032] To further illustrate these embodiments, the following examples are provided. These examples are not intended to limit the scope of the claimed invention, which should be determined solely on the basis of the attached claims.

Example 1 - Test Compounds

[0033] Rhodiola rosea, coenzyme Q_lo (also referred to herein as "CoQIO"), grape extract (from seed, pulp, and skin), alpha-lipoic acid, and quercetin were tested in human embryonic kidney cells. [0034] Coenzyme Q and grape extract alone plus the combination of all five ingredients in the ratio indicated in Table 1 (the "NRF1 blend") were also tested and cell viability and gene expression effects were measured.

TABLE 1

[0035] All test agents and combinations were dissolved or suspended in DMSO, to a concentration of 40 mg/ml. A working stock solution was made of each by diluting the 40 mg/ml stock 1 : 10 in cell culture medium, for a final concentration of 4 mg/ml in 10%

DMSO/90% medium. Appropriate dilutions were made in 96-well plates, using 10%

DMSO/90% medium as diluent, to keep DMSO concentrations constant. Final concentration of DMSO in all assays was 0.5%.

Example 2 - Cell Culture

[0036] The immortalized human embryonic kidney cell line, HEK 293, was sourced from the American Type Culture Collection (ATCC, Manassas VA, Catalog #CRL-1573). Cells were cultured in RPMI 1640 culture medium with L-glutamine (Thermo-Fisher Scientific, Waltham MA, Catalog #11875-093), supplemented with 10% fetal bovine serum and Anti- Anti antibiotic/antimycotic (Thermo-Fisher Scientific, Catalog #15240-062).

Example 3 - Cell Viability/ATP Content Assays

[0037] The CellTiter-Glo Luminescent Cell viability is a method to determine the number of viable cells in culture based on quantitation of the ATP present, as an indicator of metabolically active cells. Since ATP is made by mitochondria, this assay can be seen as a way to measure mitochondrial function. Cells were seeded at a density of 500 cells/well in 384-well white-walled Culture Plate-384 cell culture plates (Perkin-Elmer, Waltham MA, Catalog #6007680) and exposed to test agent the following day. Cells were allowed to grow in the presence of test agent for 24 hours for dose-course studies, at which point Cell-Titer- Glo (Promega Corp., Madison WI, Catalog#G7571) reagent was added at a volume equal to the cell culture medium in the plate, according to manufacturer's instructions. Luminescence was read on an Envision 2104 multilabel reader (Perkin-Elmer, Waltham WI).

Example 4 - RNA Extraction

[0038] For gene expression studies, cells were seeded at 200,000 cells/well in 6-well tissue culture plates (Genesee Scientific, San Diego CA, Catalog#25-105) and exposed to test agent the following day. At the indicated times, RNA was extracted using the Purelink RNA Mini Kit (Thermo-Fisher Scientific, Waltham MA, Catalog#12183018A). Cells were lysed on the plate using 350 of lysis buffer; otherwise, all manufacturer's instructions were followed. RNA was eluted into 35 of RNAse-free water and quantified on a NanoDrop 800 spectrophotometer (Thermo-Fisher Scientific).

Example 5 - Reverse Transcript! on/Quantitative Real-Time Polymerase

Chain Reaction (qRT-PCR)

[0039] First-strand cDNA synthesis was carried out on 1 μg of RNA, using the

Superscript VILO Master Mix (Thermo-Fisher Scientific, Catalog# 11755050) according to manufacturer's instructions. A four-fold dilution was made of each cDNA in PCR- grade water and 2 μΐ. of this solution was carried forward into qRT-PCR.

[0040] The following PCR primers were purchased from Thermo Fisher Scientific:

• MTCOl (mitochondrially encoded cytochrome c oxidase I): Hs02596864_gl (FAM label)

NRF1 : Hs01031046_ml (F AM label) PPARGClA (PGC-la): HsOl 016724 ml (FAM label) TFAM: Hs00273372_sl (FAM label)

GAPDH control: 4326317E (VIC label) [0041] Reactions were carried out in 20 total volume, made up as follows: 2 cDNA, 6 μΐ, PCR-grade water, 1 μΐ, gene-of-interest primer (FAM label), 1 μΐ, GAPDH primer (VIC label), \ 0 μΐ. Taqman Fast Advanced Master Mix (Thermo Fisher Scientific, Catalog #4444963). Reactions were run on an Applied Biosystems 7500 Fast Real-Time PCR Instrument (Thermo Fisher Scientific) under the following conditions: 50° C for 2 minutes, 95° C for 20 seconds, 40 cycles of (95° C for 3 seconds, 60° C for 30 seconds). Threshold cycle (Cr) was determined by the instrument software. Differences in threshold cycle between the gene of interest and GAPDH (ACT) were determined for each sample and used to determine fold induction of each gene of interest compared to untreated controls.

Example 6 - Cell Viability Study and Dose-Finding

[0042] Finding the dose beyond which the compounds are toxic to the cells was the first test run. This was the basis for choosing the maximal dose acceptable to run the gene expression studies. The cell viability assay was performed on each of the individual compounds (Rhodiola rosea extract, coenzyme Q₁₀, grape extract, quercetin, and alpha-lipoic acid) as well as the NRF1 blend (which contained constituent compounds at the relative proportions listed above in Table 1) at a variety of doses from 200 μg/ml down to 1.25 μg/ml. The dose to carry forward for 6 hour and 24 hour gene expression studies was defined as the maximum tolerated dose (which did not cause a significant decrease in viability) or the maximum dose at which viability was increased above 100% (as compared to untreated cells). The results from this study are shown in FIG. 1 (in FIG. 1, "NRF-1 Combo" refers to NRF1 blend).

[0043] From this study, the doses indicated in Table 2 were determined for use in the gene expression studies.

TABLE 2

Example 7 - Gene Expression Analysis

[0044] The individual compounds and the NRFl blend were evaluated for their ability to cause modulation of the expression of four genes shown to play a role in mitochondrial biogenesis and maintenance: PGC-Ια, NRFl, MTCOl, and TFAM. HEK 293 were treated for 6 hours or 24 hours with the previously-determined concentrations of each test compound, at which point RNA was extracted, subjected to first-strand cDNA synthesis (reverse transcription) and qRT-PCR for these four genes of interest, as well as GAPDH, an internal expression control. The data for PGC-Ια, NRFl, MTCOl, and TFAM are shown grouped by time point in FIGS. 2A and 2B (in FIGS. 2A and 2B, "ALA" refers to alpha-lipoic acid and "Combo" refers to NRFl blend).

[0045] At 6 hours, PGC-Ια was slightly upregulated with CoQIO at a concentration of 75 μg/ml (1.1 fold change of the relative gene expression on the y-axis) and not with any other compound or with the NRFl blend. NRFl was slightly upregulated with Rhodiola rosea (at a concentration of 10 μg/ml; 1.1 fold change). However, when the cells were treated with the NRFl blend, NRFl was almost 1.6 fold upregulated despite no upregulation for PGC-la.

[0046] 3 test agents (i.e., coenzyme Q₁₀, grape extract, and NRFl blend) were evaluated for their ability to impact cell viability and gene expression in HEK 293 cells. Specifically, these agents were chosen based on their proposed ability to modulate NRFl through upregulation of PGC-la, which may lead to increases in downstream genes such as TFAM and cytochrome c oxidase (i.e. , the four genes which were studied as part of the above- described study). Upregulation of these genes is purported to stimulate mitochondrial biogenesis and energy (ATP) production in the cell.

[0047] Both test compounds (i.e. , coenzyme Q_lo and grape extract) as well as the NRFl blend, appeared to cause an increase in either ATP production or cell viability (or both) at certain doses, driving the readout from the Cell-Titer-Glo assay above 100% when compared to controls. In terms of gene expression effects, all test agents had an inductive effect on at least one of the four genes studied at one or more doses/times, on the level of about 1.1 to about 1.6 fold. In some cases, no effect was seen on a particular gene, at one time point or the other. It is not apparent that any of these test conditions caused a significant decrease in gene expression, which has been seen for candidate compounds in past studies. Example 8 - Test for Synergistic Effects

[0048] It was evaluated whether the NRFl blend has synergistic effects on gene expression when compared to the five constituent compounds. To determine this, HEK 293 cells were treated with the individual compounds at their concentrations present in the blend (see Table 3), rather than at their maximum dose, and compared to the effect of the blend.

TABLE 3

[0049] Results after 6 hours and 24 hours of treatment are depicted in FIGS. 3A and 3B, respectively (in FIGS. 3A and 3B, "Vinovia LS Grape" refers to grape extract, "ALA" refers to alpha-lipoic acid, and "NRF-1 Combo" refers to NRFl blend). As depicted, the individual compounds at their concentrations present in the NRFl blend do not substantially activate the four genes whereas the NRFl blend exhibits a 1.6 fold activation of NRFl gene without PGC-Ια activation.

Example 9 - Global Gene Expression Analysis

[0050] The NRFl blend can be administered to groups of mice differing in age (e.g. , young versus old) to determine the effects of the NRFl blend on global gene expression in various organs of interest using RNA-Seq technology. Evaluations can be done in cardiac muscle, skeletal muscle, brain, liver, etc. As a measure of mitochondrial efficiency, the levels of Complex II (succinate dehydrogenase complex) protein can also be measured; this protein is part of the membrane-associated series of enzymes involved in mitochondrial energy production and has been shown to decrease with age.

[0051] The NRFl blend can include the components as listed and described in Table 4.

TABLE 4

R. rosea extract 220 3.67 45.83

Coenzyme Q₁₀ 100 1.67 20.83

Grape extract 70 1.17 14.58

Alpha-lipoic acid 45 0.75 9.38

Quercetin (dihydrate) 20 0.33 4.17

Total 445 7.59 94.79

*Assumes 60 kg human

⁺ Allometric dose scaling taken from FDA Guidance for Industry

(http://www fda gov/downloads/Drugs/.../Guidances/UCM078932 pdf)

Example 10 - Mouse Dosing and Sample Preparation

[0052] The NRF1 blend (see Table 4) can be tested in both young and old groups of mice as, without being bound by any one particular theory, it may be expected that mitochondrial function declines with age, and thus the effect of supplementation with the NRF1 blend (the "supplementation") may differ among these populations. To represent an aged population retired breeding stock (about 9 months of age) can be used and mice that are 3-4 weeks of age can represent a young population.

[0053] It can also be determined if any effects seen with the supplementation are dependent on dose. To address this, one group of "old" mice can be administered an elevated (e.g. , 2.5*) dose of the NRF1 blend and three mice from this group can be processed for gene/protein expression determination (this can be considered a "look-and-see" assay, as three mice may not provide statistical significance).

[0054] Upon arrival at a test facility, mice can be acclimated for three days before treatment begins. Dosing can take place on a q.d. x 7 schedule (every day, including weekends) for two weeks. 45 ICR mice, 9 per group, can be housed and treated at a vivarium, as described in Table 5.

TABLE 5

[0055] Six mice from Group 5 can continue to be dosed for an additional 2 weeks, to determine any toxic effects of high dose supplementation. The remaining three mice from this group can be processed in the same manner as the other four groups.

[0056] During the study, all mice can be weighed twice per week (e.g., on Tuesdays and Fridays) and food consumption for each group can be monitored once per week (e.g. , on Fridays). Any abnormal findings can be noted and/or communicated.

[0057] Following two weeks (i.e. , 14 total doses) of treatment, mice can be sacrificed by carbon dioxide asphyxiation, and the following organs can be collected by dissection:

• Heart (separated into two, approximately equal sections): one section can be processed for RNA-Seq (see below) and the other section can be processed for protein analysis (see below).

• Skeletal muscle: gastrocnemius (calf) muscle can be removed, flash frozen in liquid nitrogen, and then stored at -80 °C.

• Brain: the entire brain, including cerebellum and medulla oblongata, can be removed from the skull, flash frozen in liquid nitrogen, and then stored at -80 °C.

• Liver: one half of the left lateral lobe can be excised, flash frozen in liquid nitrogen, and then stored at -80 °C.

[0058] One half of each dissected heart can be processed for RNA extraction, using the Purelink RNA Mini Kit (Ambion) and using the manufacturer's recommended protocol for whole tissue. Extracted RNA from each treatment group can then be pooled into three subgroups (three mice per treatment group) and each subgroup can be taken forward into RNA-Seq as its own sample (see FIG. 4). By creating three pooled subgroups from each treatment, rather than a single pool sequenced multiple times, changes in gene expression can be more evident and statistical power can be increased. No subgroup may be generated from Group 5 (all three mice can be carried forward individually for gene expression analysis).

Example 11 - Global RNA Expression Analysis [0059] The impact of NRF1 blend on global gene expression can be assessed. This can allow alternative (and perhaps hitherto-unconceived) pathway effects to be uncovered. RNA- Seq is a technology that can allow a "snapshot" to be taken of gene expression. All RNAs present in a sample (representing all genes currently being expressed) may be sequenced and quantified to give a readout of the relative expression of each gene. By applying RNA-Seq technology to treated and untreated samples, differences in expression as a result of treatment can be elucidated, which may reveal effects unlikely to be discovered using traditional targeted gene expression analysis.

[0060] Using RNA collected from cardiac muscle tissue, as described above, RNA-Seq analysis can be performed on all five sample groups. This can reveal differences in global gene expression between young and old mice, as well as differences caused by treatment with NRF1 blend. In addition, if NRF1 blend has differential effects on young versus old populations this can also become evident.

[0061] Briefly, the RNA isolated from the cardiac muscle tissue can be run through a quality control procedure to ensure integrity, followed by ribosome depletion or poly -A selection. This process can remove ribosomes (the protein machinery which converts RNA into protein and which can interfere with downstream RNA-Seq) or can select for messenger RNA (the particular type of RNA which is converted to protein and reflects gene expression). Adaptor sequences can be ligated to the ends of each RNA, which can allow the RNA to be used in sequencing reactions and which can also provide a "barcode" to identify RNA from different samples. Following this preparation, RNA can then be sequenced (RNA-Seq) using the Illumina HiSeq platform. Fifteen samples can be subjected to RNA-Seq analysis

(representing three subgroups from each treatment Group 1 through 4 and the three individual mice from Group 5), which may require two lanes of the sequencer, to ensure > 25 million reads per sample.

Example 12 - Bioinformatics

[0062] Following RNA-Seq, sequencing data can be generated in the form of FASTQ files. The sequences generated can first be identified, by querying each sequence against the known mouse genome. At this point, the data can then be subjected to bioinformatics analysis. This can involve the creation of a number of custom algorithms, allowing raw data to be mined for such information as relative expression of each RNA, effects on known pathways, and differential effects (for example, young versus old, treated versus untreated, unique effects of treatment in aged populations, and dose-dependent changes in gene expression). This work can also involve perusal of the literature, to identify genes involved in pathways of interest and to elucidate the roles of genes which are significantly different between groups (which may provide valuable insight into the unknown or potentially off- target effects of treatment).

Example 13 - Protein Analysis for Mitochondrial Number and Function

[0063] Complex II, a mitochondria-specific collection of succinate dehydrogenase enzyme subunits, has been shown to play a role in cellular energetics. Levels of this protein have been shown to decline with age in human populations, leading to a loss of mitochondrial efficiency. This protein can be used as a marker of mitochondrial number and mitochondrial "health. " One-half of the heart material collected from each animal, as described above, can be processed and lysed for total protein extraction. TRIzol reagent can be used according to the manufacturer's instructions for animal tissue. Protein content can be quantified in each sample, using BCA reagent, to allow equal amounts of protein to be assayed in downstream assays.

[0064] Relative protein levels of the succinate dehydrogenase (SDH) complex can be measured by Western blot analysis and compared to glyceraldehyde 3-phosphate

dehydrogenase protein levels (GAPDH, a "housekeeping protein" whose expression does not generally change with treatment). Some assay development may be required, as the performance of antibodies in Western Blot can depend on the source of the protein, the manner by which it is extracted, and the equipment and reagents used. If SDH cannot be reliably and/or quickly detected, there may be a need to move to another assay strategy, such as ELISA.

Example 14 - Gene Expression Analysis at Maximal Concentrations

[0065] The maximal concentrations of the individual compounds before the individual compounds become toxic to HEK 293 cells were determined. The individual compounds at the maximal concentrations were evaluated for their ability to cause modulation of the expression of PGC-Ια, NRF1 , MTCOl, and TFAM. Results after 6 hours and 24 hours of treatment are depicted in FIGS. 5A and 5B, respectively (in FIGS. 5A and 5B, "Lipoic Acid" refers to alpha-lipoic acid).

[0066] As depicted in FIGS. 5 A and 5B, some of the individual compounds activate PGC-Ια, NRF1, MTCOl, and/or TFAM; however, the concentrations of the individual compounds are generally lower in the NRF1 blend (see, e.g. , the concentrations of alpha- lipoic acid).

Example 15 - Cell Viability Analysis

[0067] The effects of candidate compounds on cell viability at various doses were tested in HEK 293 cells treated for 24 hours. FIG. 6A is a graph showing the effect of Rhodiola rosea on cell viability at various doses in HEK 293 cells treated for 24 hours. FIG. 6B is a graph showing the effects of alpha-lipoic acid (blue) and quercetin (grey) on cell viability at various doses in HEK 293 cells treated for 24 hours.

[0068] It will be apparent to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention.

Claims

CLAIMS What is claimed is:

1. A composition comprising:

Rhodiola rosea extract; coenzyme Q₁₀; grape extract;

alpha-lipoic acid; and

quercetin.

2. The composition of claim 1, wherein the grape extract is obtained from grape seed, grape pulp, and grape skin.

3. The composition of claim 1 or 2, wherein the Rhodiola rosea extract comprises at least one of rosavins or salidroside.

4. The composition of any one of claims 1-3, wherein the alpha-lipoic acid is synthetic.

5. The composition of any one of claims 1-4, wherein a single daily dose of the composition comprises from about 25 mg to about 500 mg of Rhodiola rosea extract.

6. The composition of any one of claims 1-5, wherein a single daily dose of the composition comprises from about 10 mg to about 400 mg of coenzyme Q_lo.

7. The composition of any one of claims 1-6, wherein a single daily dose of the composition comprises from about 10 mg to about 300 mg of grape extract.

8. The composition of any one of claims 1-7, wherein a single daily dose of the composition comprises from about 5 mg to about 300 mg of alpha-lipoic acid.

9. The composition of any one of claims 1-8, wherein a single daily dose of the composition comprises from about 1 mg to about 200 mg of quercetin.

10. The composition of claim 9, wherein the quercetin is quercetin (dihydrate).

11. The composition of any one of claims 5-10, wherein the single daily dose of the composition is configured for a mammal.

12. The composition of claim 11, wherein the mammal is a human.

13. The composition of any one of claims 1-10, wherein the composition is effective for activating mitochondria when administered to a subject.

14. The composition of any one of claims 1-10, wherein the composition is effective for slowing aging when administered to a subject.

15. The composition of any one of claims 1-10, wherein the composition is effective for increasing stamina when administered to a subject.

16. The composition of any one of claims 1-10, wherein the composition is effective for increasing libido when administered to a subject.

17. A method of activating mitochondria in a subject, the method comprising: administering a therapeutically effective amount of the composition of any one of claims 1-10 to the subject.

18. A method of slowing aging in a subject, the method comprising: administering a therapeutically effective amount of the composition of any one of claims 1-10 to the subject.

19. A method of increasing stamina in a subject, the method comprising: administering a therapeutically effective amount of the composition of any one of claims 1-10 to the subject.

20. A method of increasing libido in a subject, the method comprising: administering a therapeutically effective amount of the composition of any one of claims 1-10 to the subject.

21. The method of any one of claims 17-20, wherein the subject is a human.