WO2012016018A1 - Compositions and methods for improvement of oxygen metabolism and cytokine regulation - Google Patents

Abstract

Compositions and methods for dietary supplements that contain a water-extractable fraction of a complex fermentation product are presented that are effective to modulate in vivo cytokines, oxygen consumption rate, extracellular acidification rate, and intracellular ATP production. Thus, contemplated compositions are particularly desirable in reducing symptoms associated with compromised oxygen utilization in a person.

Description

COMPOSITIONS AND METHODS FOR IMPROVEMENT OF OXYGEN METABOLISM AND CYTOKINE REGULATION

[0001] This application claims priority to our copending US provisional applications with the serial numbers 61/368542, filed July 28, 2010, and 61/425180, filed December 20, 2010, both of which are incorporated by reference herein.

Field of the Invention

[0002] The field of the invention is compounds, compositions, and methods for improvement of oxygen and energy metabolism, especially as it relates to functional foods and nutritional supplements.

Background of the Invention

[0003] Energy metabolism is closely linked to oxygen utilization, which is best illustrated when comparing the energy balances of aerobic and anaerobic glucose utilization. Under aerobic oxidative conditions, glucose is completely metabolized to C02 and H20, yielding 34 mol ATP/mol glucose, whereas under anaerobic, oxygen depleted conditions, glucose is incompletely metabolized to lactic acid yielding only 2 mol ATP/mol glucose.

[0004] Unfortunately, there are numerous conditions and circumstances under which organs and tissues can experience oxygen partial pressures that are less than ideal. Moreover, there are also various conditions and circumstances under which organs and tissues are not fully capable of utilizing oxygen, even when oxygen is present at relatively high partial pressure. To help improve oxygen utilization in an organism, supplemental oxygen can be provided via inhalation. However, such supplementation is often impractical. Reduced or impaired oxygen utilization is experienced in a variety of sign and symptoms, and typically adversely affects exercise capacity, strength and stamina, and mental activity. Moreover, reduced oxygen utilization may also lead to less than ideal catabolism of energy substrates, which in turn, can lead to increased body mass and reduced capability of losing excess body mass. Still further, reduced oxygen utilization may also lead to a drop in subjective energy levels and an increase in fatigue.

[0005] Consequently, it would be desirable to have a modality that effectively increases oxygen utilization in a manner that can be easily and conveniently administered without the need for respiratory supplemental oxygen. Summary of The Invention

[0006] The inventors have discovered that certain nutritionally acceptable compositions are effective to improve various aspects of oxygen utilization, energy production, and cytokine expression, and that such compositions can be prepared in a relatively simple and effective manner.

[0007] In one especially preferred aspect of the inventive subject matter, a dietary supplement includes a water-extractable fraction of a complex fermentation product together with a nutritionally acceptable carrier, wherein the complex fermentation product is a fermented mixture of a sprout, whey protein, an algae, and one or more nutritionally acceptable enzymes. It is generally preferred that the water-extractable fraction is present in the nutritional supplement in amount effective to provide one or more desirable effects on oxygen utilization, energy production, and cytokine modulation. Most preferably, contemplated supplements will therefore upon oral administration increase in vivo interleukin levels (and especially IL-1, IL-10, IL-12, and/or IL-15), and/or oxygen consumption rate, and/or decrease extracellular acidification rate. Thus, contemplated compositions will also increase the ratio of oxygen consumption rate to extracellular acidification rate, and/or intracellular ATP production.

[0008] It is still further preferred that the sprout is a sprout of a Brassicaceae plant or a sprout of a Fabaceae plant, that the algae is Aphanizomenon flos-aquae, Chlorella spec, Fucus vesiculosus, Gracilaria spec, Porphyra spec, Saccharina japonica, or Arthrospira spec.(Spirulina), and/or that the enzyme is a protease, an amylase, a cellulase, bromelain, papain, and/or lactase. While not limiting to the inventive subject mater, it is generally preferred that the complex fermentation product is a short-term fermentation product (e.g., fermented for between about 12-36 hours), and/or that the nutritionally acceptable carrier is a liquid carrier. In still further preferred aspects, the water-extractable fraction is a concentrate of a water extract of the complex fermentation product or a fraction that is obtained by chromatography of the water extract of the complex fermentation product.

[0009] With respect to desirable quantities, it is generally contemplated that all quantities are suitable that provide a measurable effect in vivo upon oral administration. Consequently, suitable quantities are those that are effective to increase one or more interleukins, that increase oxygen consumption rate, decrease extracellular acidification rate, increase a ratio of oxygen consumption rate to extracellular acidification rate, and that increase the intracellular ATP production. For example, the water-extractable fraction of the complex fermentation product may be present in the dietary supplement in an amount of at least 5 wt%.

[0010] Consequently, the inventors also contemplate a method of modulating energy metabolism and/or cytokine production in a mammal, in which in one step a water- extractable fraction of a complex fermentation product is provided, wherein the complex fermentation product is a fermented mixture of a sprout, whey protein, an algae, and a plurality of nutritionally acceptable enzymes. In another step, the water-extractable fraction is combined with a nutritionally acceptable carrier in an amount effective to increase an interleukin selected from the group consisting of IL-1, IL-10, IL-12, and IL-15, to increase oxygen consumption rate, to decrease extracellular acidification rate, to increase a ratio of oxygen consumption rate to extracellular acidification rate, and/or to increase the intracellular ATP production, to thereby produce a nutritional supplement. In still another step, the nutritional supplement is provided to the mammal in an amount effective to modulate the energy metabolism and/or cytokine production in the mammal.

[0011] With respect to the sprout, the algae, the enzyme, and the fermentation time, the same considerations as provided above apply. In particularly preferred aspects, modulation of the energy metabolism or cytokine production in the mammal is effective to increase exercise capacity or strength, to reduce adverse effects due to rapid altitude change, to increase memory function, and/or to alleviate a symptom associated with a respiratory condition.

[0012] Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

Brief Description of The Drawing

[0013] Figures 1A and IB are graphs exemplarily depicting dose-dependent oxygen usage of cells treated with compositions according to the inventive subject matter.

[0014] Figure 2 is a graphical representation of acute and late OCR/ECAR effects in cells in response to treatment with compositions according to the inventive subject matter.

[0015] Figures 3A and 3B are graphs depicting respective acute and late OCR/ECAR effects in cells in response to treatment with compositions according to the inventive subject matter. [0016] Figure 4A is a graph depicting intracellular and extracellular ATP production of cells in response to treatment with compositions according to the inventive subject matter.

[0017] Figure 4B is a table listing lactic acid production of cells in response to treatment with compositions according to the inventive subject matter.

[0018] Figure 5 is a graph of OCR/ECAR effects in cells in response to treatment with compositions according to the inventive subject matter.

[0019] Figure 6 is a graph depicting intracellular and extracellular lactic acid of cells in response to treatment with compositions according to the inventive subject matter.

[0020] Figure 7 is a graph depicting change in VAS score in volunteers treated with compositions according to the inventive subject matter.

[0021] Figure 8 is a table listing change in cytokine/chemokine production in peripheral blood cells of human volunteers treated with compositions according to the inventive subject matter.

Detailed Description

[0022] The inventors have discovered that various nutritionally acceptable compositions and methods not only significantly improve oxygen utilization in a mammal, but also modulate cytokine and chemokine profiles in vivo. Preferably, compositions contemplated herein are orally administered in a nutritionally acceptable liquid or solid carrier. Among other desirable effects, compositions and methods presented herein will significantly increase oxygenation of blood in vivo, increase intracellular ATP levels in vivo, increase oxygen consumption rate (OCR) in vivo, decrease extracellular acidification rate (ECAR), substantially increase the ratio of OCR/ECAR, and stimulate anti-inflammatory cytokines.

[0023] Based on the below data and additional considerations, the inventors contemplate that compositions and methods presented herein may be useful in modulating energy metabolism in a mammal. Consequently, it is also contemplated that the compositions and methods herein are useful in producing an increase in OCR, a decrease in ECAR, an increase in the ratio of OCR/ECAR, an increase in intracellular ATP levels, and/or an increase in intracellular oxygen utilization. Viewed from a different perspective, it should be appreciated that the compositions and methods presented herein allow for more efficient use of existing oxygen from the cellular milieu, and/or a mode in which cells are activated to utilize more oxygen.

[0024] Therefore, it is contemplated that the compositions according to the inventive subject matter will be useful in the treatment of various signs and symptoms of conditions associated with a reduction in oxygen utilization. For example, it is contemplated that the compositions may be useful in the treatment (e.g., to provide symptomatic relief or subjective well being, or to increase OCR, decrease ECAR, etc.) of adverse effects due to rapid altitude change (e.g., change in altitude of at least 2000 ft in less than 24 hrs), or of symptoms of a respiratory condition (e.g., due to chronic obstructive pulmonary disease, bronchitis, asthma, emphysema, tobacco use, autoimmune disorder, obesity, air pollution, etc.). Contemplated compositions may be especially useful in increasing cerebral oxygen utilization and thus be suitable to increase memory function (e.g., executive functions, cognitive functions, etc.). Still further, contemplated compositions may also be advantageous in restoration of and/or increase of exercise capacity in sports or in daily activities, increase in V02 max (maximal oxygen consumption, aerobic capacity), and endurance, as well as in a delay in onset of anaerobic metabolism under strain.

[0025] It should still further be appreciated that contemplated compositions and methods increase the intracellular ATP concentration without substantial generation (i.e., less than 10%, and more typically less than 5% as compared to pre-administration) of various radical species, and especially without increased production of ROS and HORAC (data not shown). Such finding is particularly noteworthy as an increase in endogenous (intracellular) ATP is typically associated with an increase in intracellular free radicals. In this context, it should be appreciated that cells function differently at high intracellular ATP levels without free radicals as compared to low levels of intracellular ATP. In case of blood cells such difference is particularly important (e.g., different activities of lymphocytes form multiple sclerosis patients due to different intracellular ATP concentration). Therefore, especially contemplated uses also include those where an increase in intracellular respiration activity would be particularly desirable or beneficial (e.g., muscle performance, delay of aging, MS, cancer, skin aging/senescence, hypoxia, ischemia, cardiovascular, autoimmune, immunological responsiveness, metabolism including appetite control and CNS function, and others).

Likewise, especially contemplated uses also include those where conditions related to reduced oxygen levels in blood are encountered (e.g., systemic depressed respiration, COPD, asthma, Bronchitis, etc.)

[0026] Additionally, and especially where individual compounds ('actives') are isolated from the compositions presented herein, such individual compounds may be administered in various medical uses, and especially in surgical interventions where hypoxia is commonly encountered (e.g., open heart surgery, organ transport and/or transplantation, induced hypothermia after traumatic brain injury, etc.).

[0027] Particularly preferred compositions and methods are exemplarily provided in the experimental section below. However, it should be appreciated that numerous modifications may be made to the compositions and methods without departing from the inventive concept presented herein. Consequently, a method of modulating energy metabolism in a mammal (e.g., human, but also a pet, a farm animal, or a zoo animal) is generally contemplated in which in one step at least one of a plant material, a milk-based material, an algae-based material, and a plurality of enzymes is provided. In another step, the material(s) is/are then combined with a first solvent to form a fermentation mix, and fermentation is allowed to proceed (typically aerobic fermentation at room temperature [20-22 °C]) for a predetermined period of time, typically at least 6 hours. In yet another step, the fermentation mix is then extracted with a second solvent after the predetermined period of time in order to so form an extract, that is then provided (directly or after further processing) to the mammal in an amount effective to modulate energy metabolism in the mammal.

[0028] Most typically, the plant material is a monocot (e.g., various grasses) or dicot (e.g., various cruciferous plants) material, preferably as a sprout material, the milk-based material is whey powder or liquid, the algae-based material is spirulina-based (most preferably spirulina powder), and the enzymes are enzymes suitable for nutritional supplementation (e.g., proteases, lipases, amylase, glycosidases, etc.). However, numerous alternative plant materials are also contemplated and include fruits, roots, leaves, and seeds, each of which may be extracted using one or more solvents. Similarly, the milk-based material may comprise a protein fraction (e.g., lactalbumin, lactoferrin, etc.), a saccharide fraction, etc, and the algae based material may include all edible algae formulations (as powder or liquid, optionally processed to enrich one or more ingredients). Additionally, contemplated ingredients may also include various fungal-based materials (e.g., Reishi Maitake Shiitake, etc). [0029] For example, especially suitable sprouts will be cruciferous sprouts (i.e., a sprout of a Brassicaceae plant) and/or a sprout of a Fabaceae plant (and especially alfalfa sprout). However, numerous other plant materials are also deemed suitable for use herein and will generally include all nutritionally acceptable plants (e.g., root vegetables, berries, etc).

Similarly, there are numerous nutritionally acceptable algae known in the art, and all of those are considered appropriate for use herein. Thus, especially preferred algae include

Aphanizomenon flos-aquae, Chlorella spec., Fucus vesiculosus, Gracilaria spec., Porphyra spec., Saccharina japonica, and Arthrospira spec. (Spirutina). Most typically, the algae will be provided as a dry material (e.g., powdered whole cells or powdered lysed cells), which may or may not have been subjected to further purification steps (e.g., removal of insoluble materials, solvent extraction with aqueous or ethanolic solvent, etc.). With respect to suitable enzymes it is contemplated that all nutritionally acceptable enzymes are suitable for use herein. However, especially preferred enzymes include numerous proteases, amylase, cellulase, bromelain, papain, and lactase.

[0030] Most typically, fermentation is performed in an aqueous solvent (and most preferably in water) using one or more of the enzymes provided above. Thus, it should be appreciated that in at least some aspects of the inventive subject matter, the fermentation will be a non- microbial enzymatic fermentation. However, in less preferred aspects, fermentation may also involve microbial fermentation using bacterial (e.g., lactobacilli) and/or yeast cells (e.g., saccharomyces). Moreover, it should be appreciated that the fermentation is preferably carried out under aerobic conditions as further discussed below, however, anaerobic fermentation is also deemed suitable. As is discussed in more detail below, it is generally preferred that the fermentation is performed at about room temperature (between 20 and 30 °C). However, in alternative aspects, higher temperatures (e.g., 30-37 °C, and even higher) or lower temperatures (e.g., 10-20 °C, and even higher) are also contemplated herein. With respect to the duration of the fermentation it is generally preferred that the duration is between 12 and 60 hours (to so present a short-term fermentation product), and more preferably between 18-48 hours. However, in at least some aspects, longer fermentation durations (e.g., between 60 and 72 hours, and even longer) are also deemed appropriate.

[0031] The inventors have observed that the length of fermentation time can significantly alter the composition of the ferment and with that the biological effect of the resultant fermented materials (e.g., the observed OCR/ECAR of a fermentation after 96 hours is significantly different from OCR/ECAR of a fermentation after 24 hours). Consequently, the inventors contemplate that various lengths of fermentation may produce materials with differing but still desirable biological activities. If needed, the fermentation can be performed at relatively low temperatures (e.g., between 4 and 20 °C) for longer periods, or at elevated temperatures (e.g., between 25 and 60 °C), typically for a relatively short time. Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

[0032] Once finished, the fermentation product is then extracted with the second solvent, which may be at least partially removed to obtain a more concentrated extract. Regardless of the solvent, the extract may also be further processed by chromatographical methods (e.g., anion exchange chromatography, cation exchange chromatography, molecular size exclusion chromatography, affinity chromatography, etc.), but chemical methods (e.g., reaction with one or more reagents, preferably nutritionally acceptable reagents), thermal processing (e.g., drying, baking, etc.), and so on. Where the fermentation is performed in a relatively large volume, it should be noted that the complex fermentation product may already be in an aqueous solvent. Alternatively, and especially where the fermentation is performed in a relatively small volume of solvent, the complex fermentation product may be extracted with an aqueous solvent (e.g., solvent with a water content of at least 5%, most typically at least 50%). Therefore, the term "water-extractable fraction of the complex fermentation product" will include both, the aqueous fermentation medium as well as an aqueous extract of a complex fermentation product. The extraction temperature will typically be at a temperature of between 10 °C and 95 °C, and most preferably between 20-35 °C. Suitable co-solvents especially include nutritionally acceptable solvents, which are well known in the art. Where needed, further solvents other than aqueous solvents may also be employed.

[0033] Regardless of the manner of preparation of the water-extractable fraction, it is generally preferred that the water-extractable fraction of a complex fermentation product is combined (after optional processing) with a nutritionally acceptable carrier to so form a dietary supplement. For example, suitable liquid carriers include water, tea, coffee, fruit juices, etc. Suitable solid carriers include snack bars, cereal and cereal products, dairy product, backed goods, etc. Depending on the particular use, it should be appreciated that the water-extractable fraction of the complex fermentation product may be present in the dietary supplement in varying amounts. However, it is generally preferred that the water-extractable fraction is present in an amount of at least 0.01 wt%, more typically at least 0.1 wt%, even more typically at least 1 wt%, and most typically at least 5 wt%. Thus, and viewed from a different perspective, the water-extractable fraction may be present in the nutritional supplement in amount effective to increase an interleukin, increase oxygen consumption rate, decrease extracellular acidification rate, increase a ratio of oxygen consumption rate to extracellular acidification rate, and/or to increase the intracellular ATP production.

Experimental Data

[0034] The following examples are provided to give exemplary guidance as to how to make and use the compositions according to the inventive subject matter.

[0035] Composition I was prepared as follows: 50 g of a fermented blend (fermentation time 16 days) of alfalfa sprout powder, whey protein, spirulina powder, and an enzyme mixture (protease, amylase, cellulase, bromelain, papain, lactase) were combined with 730 ml of water, mixed thoroughly to hydrate/wet the entire sample. The so prepared mixture was covered loosely to permit airflow and to provide protection of the mixture from particulate matter in ambient air. The mixture was allowed to stand at 70 - 78 °F for 1 hour.

[0036] A French coffee press was then used to remove excess fibrous mass to provide an estimated yield of about 500 ml supernatant. Insoluble materials were removed as needed by centrifugation and/or ultrafiltration. The cleared solution was then tested as further described below.

[0037] Composition II was prepared as follows: 50 g of a alfalfa sprout powder, 730 ml of whey liquid, 2.1 g of spirulina powder, and 1.8 g of enzyme mixture (protease, amylase, cellulase, bromelain, papain, lactase) were mixed thoroughly. The so prepared mixture was covered loosely to permit airflow and to provide protection of the mixture from particulate matter in ambient air. The mixture was allowed to stand at 70 - 78 °F for 1 hour, and then to ferment at ambient temperature (70 - 78 °F) to several predetermined time points (T = 24, 48, 72, 96 hrs.). The mixture was stirred daily following the first day to ensure uniformity of the biomass and uniform fermentation. [0038] A French coffee press was then used to remove excess fibrous mass to provide an estimated yield of about 500 ml supernatant. Insoluble materials were removed as needed by centrifugation and/or ultrafiltration. The cleared solution was then tested as further described below.

[0039] Composition III was an aqueous dilution of Composition I. Composition III was also partially evaporated to form a concentrate. The solutions were then tested as further described below.

[0040] Composition IV was prepared as follows: 50 g of a blend (fermentation time 16 days) of alfalfa sprout powder, whey protein, spirulina powder, and an enzyme mixture (protease, amylase, cellulase, bromelain, papain, lactase) were combined with 70 ml of water, mixed thoroughly to hydrate/wet the entire sample. The so prepared mixture was covered loosely to permit airflow and to provide protection of the mixture from particulate matter in ambient air. The mixture was allowed to stand at 70 - 78 °F for 1 hour, and was subsequently fermented for 16 days. Water was added as needed to keep the mixture moist. At the end of the fermentation, 700 ml water was added and thoroughly mixed.

[0041] A French coffee press was then used to remove excess fibrous mass to provide an estimated yield of about 500 ml supernatant. Insoluble materials were removed as needed by centrifugation and/or ultrafiltration. The cleared solution was then tested as further described below.

[0042] Composition V was prepared as follows: 50 g of a alfalfa sprout powder, 70 ml of whey liquid, 2.1 g of spirulina powder, and 1.8 g of enzyme mixture (protease, amylase, cellulase, bromelain, papain, lactase) were mixed thoroughly. The so prepared mixture was covered loosely to permit airflow and to provide protection of the mixture from particulate matter in ambient air. The mixture was allowed to stand at 70 - 78 °F for 1 hour, and was subsequently fermented for 16 days. Water was added as needed to keep the mixture moist. At the end of the fermentation, 700 ml water was added and thoroughly mixed.

[0043] A French coffee press was then used to remove excess fibrous mass to provide an estimated yield of about 500 ml supernatant. Insoluble materials were removed as needed by centrifugation and/or ultrafiltration. The cleared solution was then tested as further described below. [0044] Oxygen utilization of cells in vitro: Composition III was used in original strength and in concentrated form after 70% evaporation of water in a commercially available test format (Luxcel Biosciences, catalog number: MitoXpress-Xtra-KI) following the manufacturer's recommendations. More specifically, the MitoExpress assay was used in this study to monitor oxygen utilization by treated cells. This assay is based on oxygen-sensitive fluorescent probe. At high concentration of oxygen in water phase, fluorescent signal of the probe is measured by Relative Fluorescent Units (RFU). When oxygen is utilized, RFU is increasing. In this experimental set up, cultivated cells were treated with Composition III or water as control at indicated doses. RFU were measured during 1 hour. Presented data are average measurements collected after 1 hour of exposure to Composition III. Cells were cultivated in 96-well plate for 24 hrs, washed, and exposed to fresh medium (control) or medium supplemented with Composition III (neat and concentrated) at dilutions as indicated in Figure 1A.

[0045] In a second set of experiments, peripheral blood cells were isolated from healthy subjects using CPT tubes. The procedure was provided by the provider (BD Inc.). After isolation, 2 million cells were suspended in culture buffer with or without Composition III. Oxygen utilization (RFU) was followed for 60 minutes. Presented data reflect oxygen utilized after one hour of treatment with Composition III as shown in Figure IB.

[0046] As can be seen from Figures A and B, all data collected indicated that Composition III stimulates utilization of oxygen by cells in vitro in a dose-dependent manner under the described experimental conditions. As shown in Figure A, concentrated Composition III increased oxygen utilization by Hepa lc liver cells up to 65 % at dose 0.1%. Under the same conditions and at the same dose, the neat Composition III had shown a similar, although less, stimulatory effect on oxygen utilization of up to 24%. In comparison, commercial water purchased from Sigma Aldrich (Sigma Water) failed to show increased oxygen utilization under the same experimental conditions. Further experimental work (data not shown) indicated that concentrated Composition III increases oxygen utilization in human peripheral blood cells that had been freshly collected from healthy human subjects. As presented in Fig 2, concentrated Composition III at dose 0.1% increases oxygen utilization up to 22% after the first hour of the treatment. At dose 0.2%, concentrated Composition III increases oxygen utilization up to 34% under the same experimental conditions. These results show that the stimulatory effect of Composition III on oxygen utilization is not limited to liver cells Hepa lc, an established cancer cell line. Therefore Composition III is deemed suitable for stimulation of oxygen utilization in normal human cells.

[0047] It is of importance to note that the activity of concentrated Composition III described above is more significant than the effect of neat Composition III. This clearly demonstrates that the activity is likely due to chemical entities present in the water that have been produced during the production processes. Consequently, it is contemplated that the chemical composition of the small molecules within Composition III determines the stimulatory effect of this product on oxygen utilization by treated cells in vitro. Another interesting observation was that Composition III enhances oxygen utilization of the exposed cells with some delay rather than immediately. This strongly indicates that Composition III induces oxygen utilization in an indirect manner.

[0048] Mitochondrial activity of contemplated and exemplary compositions: The following experiments were performed to establish stimulatory effect of contemplated compositions on oxygen metabolism in human cells as measured by OCR (Oxygen Consumption Rate) and ECAR (Extracellular Acidification Rate). Testing was performed using Seahorse technology in order to measure real-time changes in oxygen consumption by freshly isolated human peripheral blood cells. Additionally, the same technology was employed to measure acidification rate, (an indicator of lactic acid production by cells treated with tested samples). Increases in extracellular level of lactic acid production under these experimental conditions are correlated with increased glucose utilization via glycolysis rather than oxidative phosphorylation.

[0049] More specifically, to increase accuracy of testing five wells were used for each condition (untreated control, 10% of Sigma water, or 10% of tested sample). Each experimental data point represents an average of results collected from 5 "read-outs" for each experimental time point. All results were generated on freshly isolated blood cells from different and randomly selected healthy and fasted donors. Internal Controls: FCCP and oligomycin, two common drugs used in research on mitochondria, were used in each experiment to confirm accuracy of testing. FCCP stimulate cells to use more oxygen;

however, glycolysis remained the main process to generate ATP and energy. Therefore, treatment of cells with FCCP results in high values for OCR and ECAR. By average, FCCP caused 397% increase in OCR and treatment with oligomycin resulted by average 86% inhibition of OCR. These two values were used as positive and negative controls, respectively, in order to ensure quality of test, viability and metabolic functionality of fresh human blood cells. Oligomycin inhibits oxygen metabolism. In presence of this substance, cells generate ATP/energy mainly via glycolysis. Therefore, treatment of cells with oligomycin under these experimental conditions resulted in high ECAR values. To ensure quality of human blood cells, blood was collected directly into Cell Preparation Tube with Sodium Citrate, Becton Dickinson Inc. Peripheral blood cells were isolated during 45 minutes following Protocol provided by Becton Dickinson. Blood cells were cultivated during each experiment in Seahorse media supplemented with 5mM of glucose. Glucose concentration was adjusted from 1 1 mM to 5 mM to avoid testing of samples under hyperglycemic conditions. All results presented here were generated in presence of glucose at concentration lower than 95mg/dl.

[0050] The following samples were used for the above series of experiments: Set 1, samples S1-S8 (selected samples from Composition I as described above), Set 2, Samples S9-S 16 (selected samples from Composition II as described above at time points 24, 48, 72, and 96 hrs), and Set 3, samples S19-S26 (selected samples from Composition IV as described above). Sample 3 A: Evolve water, neat (commercially available); 3C: Evolve, concentrated by 70% (70% of water was slowly evaporated); 6A: Composition III; 6C: Composition III (concentrated by 70%); 7A: Alexa water, neat (commercially available); 7C: Alexa water, concentrated by 70% (70% of water was slowly evaporated). All samples were in liquid form, 50mL tube each. This material was used as 100% sample, and diluted 10 times in

experimental media to provide final concentration 10%. All samples were filtered by a 0.5 micron filter and pH adjusted to 7.4 value as required by Seahorse Protocol to perform OCR and ECAR tests.

[0051] Results: In order to simplify reporting of these complex data, an "OCR/ECAR index" is presented herein. OCR/ECAR index is a single number generated by dividing OCR% increase or decrease compared to Sigma Water control by the ECAR% change over the same control. Consequently, samples showing an OCR/ECAR index > 100% indicate that they more are capable of induction of oxygen metabolism rather than anaerobic glycolysis. For comparison, those samples showing this index value < 100% indicate that said samples preferably stimulate anaerobic glycolysis over oxygen metabolism and oxygen-dependent stimulation of energy. Also, samples with index value >100 may provide efficient anti- hypoxic effect. OCR/ECAR Index data are presented in two Groups (Figure 2): Immediate/acute effect of tested samples during first 10 minutes after injection (First bar of pair of bars), and late effect measured at 90 minutes following injection of tested sample into wells with human fresh blood cells (Second bar of pair of bars). Short horizontal lines indicate average value of OCR/ECAR Index for each set of samples, and long bottom horizontal line indicates zero-change point, based upon water control value.

[0052] Figure 3A illustrates in a different graphical format the distribution of acute OCR% and acute ECAR% values, and Figure 3B illustrates in a different graphical format the distribution of late OCR% and late ECAR% values. These graphs illustrate that several samples are located in upper left part of the graph, indicating increased oxygen metabolism, (above the 100% OCR value which is Sigma water control value), and reduced glycolysis processes which result in extracellular acidification due to production of lactic acid (less than 100% of ECAR). Interestingly, several samples are showing significant effect during first 10 minutes of the treatment (Figure 3 A, acute effect). This trend is more obvious as presented on Figure 3B where more ECAR values are shifted to range 60-80% over untreated control.

[0053] Altogether, at this time there is a remaining set of results required in order to show level of ATP production upon treatment with contemplated compositions, especially the sample set S9-S16. Thus far, samples 10 and 16 were tested for ATP, showing multifold increase in ATP production and with minor increase in lactic acid production. Sample S10 was found more potent in stimulation of ATP than sample S 16.

[0054] Consequently, it should be appreciated that none of tested samples provided inhibitory effect on oxygen metabolism. Moreover, the data also show that samples S 1-S8 were active in the range 4-21% during first 10 minutes of the treatment, and 18-53% after 90 minutes of the treatment. Samples S9-S16 provided significant activity, and samples S9-S14 caused over 100% (doubling) increase in value of OCR/ECAR Index during first 10 minutes of the treatment. Interestingly, induction of oxygen metabolism is further maintained after 90 minutes of the treatment. Samples S15-S 16, on the other hand show less stimulatory effect during first 10 minutes, however, 90 minutes treatment resulted in up to 70% increase in OCR ECAR Index value over control (media with 10% Sigma water. Overall, Samples SI 5- S16 were found highly active but at significantly lower level than samples S9-S14. In a third set of samples, only sample S25 and S26 were found active in range up to 100 % (doubling) but only after 90 minutes of the treatment. Overall, average increase in OCR/ECAR Index for samples 19-26 is much lower than in case of samples S9-S16. [0055] Samples 3 A and 3C caused increase in OCR/ECAR Index value by 1 15% after 90 minutes. In comparison, Sample 3C caused 118% increase after the same period of time. This is not considered as different effect of 3 A versus 3C. However, following acute effect, treatment with 3A resulted in 105% increase of OCR/ECAR Index whereas 3C caused acute 1 18% increase. Therefore, 3C sample is initially more acutely potent than 3A but not during first 90 minutes of the treatment. Two other waters used in this testing and reported here, 6A/6C and 7A/7C are less active than sample 3A/3C.

[0056] Based on the above results, a second batch of samples (Batch #2) was prepared following the preparation of Composition II and samples were collected after 1, 4, 24, 48, 72 and 96 hours of fermentation (reported here as P I, P4, P24, P48, P72 and P96, respectively). These samples were tested at 1% concentration. Results collected from Batch #2 samples showed that P24 and P48 were the most active for intracellular ATP (iATP) level changes and OCR/ECAR changes, respectively. Interestingly, very low concentrations of extracellular ATP (eATP) were also observed. As presented in Figure 4A, P24 stimulated iATP generation to the highest level. Under the same conditions extracellular ATP (eATP) remained low. This result confirmed that high concentrations of ATP measured were definitely generated and present inside treated cells. During this phase of testing it became clear that samples were getting progressively enriched in extracellular lactic acid (eLA) during fermentation (see Figure 4B). Figure 5 depicts OCR/ECAR [% over H20 Control]. Higher OCR/ECAR value indicates higher oxygen consumption at lower cell acidification (lactate generation). Following this parameter, sample P48 provides the highest OCR/ECAR ratio value

[0057] The inventors also measured intracellular Lactic Acid (iLA) during treatment by Batch #2 samples, (results are presented in Figure 6). Interestingly, iLA was increased by treatment with all samples (PI - P96). This indicated that any observed changes in iATP upon treatment by the P samples were even more significant. Moreover, amounts of iLA was in nanoMolar concentrations/well, whereas iATP concentrations were at a hundreds-of-times- higher range ^grams/well). This observation strongly supports the case that these samples indeed increase oxygen utilization, resulting in favorable increases of ATP generation over LA generation. Thus, sample P24 seems to be the most active in generation of intracellular ATP and stimulation of oxygen utilization. Sample P48 was found even more active by OCR/ECAR ratio; however, its iATP profile was less favorable than in P24. [0058] In still further aspects of the inventive subject matter, the inventors also surprisingly discovered that the compositions according to the inventive subject matter are effective to modulate cytokine levels in a human. For example, in a controlled acute study involving human volunteers a significant dose-dependent effect was observed using 100 mg, 400 mg, and 1200 mg administration of an extract as described in the appendices and above. The study used fasted subjects with three healthy volunteers per group receiving a single dose treatment. Experimental time-points were 0, 30, 60, 90, 120 and 180 minutes after oral administration, and cytokines were measured in collected sera by standard Elisa assays. Follow-up procedures included blood chemistry analysis, determination of cytokine and chemokine profiles in collected sera, and VAS (visual analog scale) evaluation. Adverse effects were not reported, and blood chemistry data did not show any unfavorable changes due to the treatments, even with highest dose of 1200 mg. Liver enzymes (AST, ALT, LDH, BUN, GGTP), mineral profile, and blood glucose, and triglycerides were not changed.

[0059] Results of the VAS data are shown in Figure 7 in which Ql is Energy level in the morning, Q2 is Intensity of fatigue in the morning, Q3 is Intensity of headache in the morning, Q4 is Intensity of facial pain-pressure, Q5 is Level of appetite in the morning, and Q6 is Level of thirst in the morning. Figure 8 illustrates exemplary changes in cytokine and chemokine levels that were observed in the above study group. Here, it is readily apparent that contemplated compounds and compositions are already immunologically active at a dose of 100 mg. Therefore, daily dosage of contemplated compounds and compositions will preferably be in a range of between 10 mg and 1000 mg, and more preferably between 50 mg and 500 mg, and most preferably between 80 mg and 250 mg.

[0060] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C .... and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

What is claimed is:

|. A dietary supplement comprising:

a water-extractable fraction of a complex fermentation product in combination with a nutritionally acceptable carrier;

wherein the complex fermentation product is a fermented mixture of a sprout, whey protein, an algae, and a plurality of nutritionally acceptable enzymes; and wherein the water-extractable fraction is present in the nutritional supplement in amount effective to at least one of (a) increase an interleukin selected from the group consisting of IL-1, IL-10, IL-12, and IL-15, (b) increase oxygen consumption rate, (c) decrease extracellular acidification rate, (d) increase a ratio of oxygen consumption rate to extracellular acidification rate, and (e) increase the intracellular ATP production.

2. The dietary supplement of claim 1 wherein the sprout is selected from the group consisting of a sprout of a Brassicaceae plant and a sprout of a Fabaceae plant.

3. The dietary supplement of claim 1 wherein the algae is selected from the group

consisting of Aphanizomenon flos-aquae, Chlorella spec, Fucus vesiculosus, Gracilaria spec, Porphyra spec, Saccharina japonica, and Arthrospira

spec. (Spirulina) .

4. The dietary supplement of claim 1 wherein the enzyme is selected from the group consisting of a protease, an amylase, a cellulase, bromelain, papain, and lactase.

5. The dietary supplement of claim 1 wherein the complex fermentation product is a short-term fermentation product.

6. The dietary supplement of claim 1 wherein the nutritionally acceptable carrier is a liquid carrier.

7. The dietary supplement of claim 1 wherein the water-extractable fraction is a

concentrate of a water extract of the complex fermentation product.

8. The dietary supplement of claim 1 wherein the water-extractable fraction is a fraction obtained by chromatography of a water extract of the complex fermentation product.

9. The dietary supplement of claim 1 wherein the water-extractable fraction of the complex fermentation product is present in the dietary supplement in an amount of at least 5 wt%.

10. The dietary supplement of claim 1 wherein the water-extractable fraction is present in the nutritional supplement in amount effective to increase an interleukin, increase oxygen consumption rate, decrease extracellular acidification rate, increase a ratio of oxygen consumption rate to extracellular acidification rate, and to increase the intracellular ATP production.

1 1 . A method of modulating at least one of energy metabolism and cytokine production in a mammal, comprising:

providing a water-extractable fraction of a complex fermentation product, wherein the complex fermentation product is a fermented mixture of a sprout, whey protein, an algae, and a plurality of nutritionally acceptable enzymes;

combining the water-extractable fraction with a nutritionally acceptable carrier in an amount effective to at least one of (a) increase an interleukin selected from the group consisting of IL-1, IL-10, IL-12, and IL-15, (b) increase oxygen consumption rate, (c) decrease extracellular acidification rate, (d) increase a ratio of oxygen consumption rate to extracellular acidification rate, and (e) increase the intracellular ATP production, to thereby produce a nutritional supplement; and

providing the nutritional supplement to the mammal in an amount effective to

modulate at least one of energy metabolism and cytokine production in the mammal.

12. The method of claim 11 wherein the sprout is selected from the group consisting of a sprout of a Brassicaceae plant and a sprout of a Fabaceae plant.

13. The method of claim 1 1 wherein the algae is selected from the group consisting of

Aphanizomenon flos-aquae, Chlorella spec, Fucus vesiculosus, Gracilaria spec, Porphyra spec, Saccharina japonica, and Arthrospira spec. (SpiruUna).

14. The method of claim 1 1 wherein the enzyme is selected from the group consisting of a protease, an amylase, a cellulase, bromelain, papain, and lactase. The method of claim 1 1 wherein the complex fermentation product is a short-term fermentation product.

The method of claim 1 1 wherein the nutritionally acceptable carrier is a liquid carrier.

The method of claim 1 1 wherein the modulation of the energy metabolism or cytokine production in the mammal is effective to increasing exercise capacity or strength.

The method of claim 1 1 wherein the modulation of the energy metabolism or cytokine production in the mammal is effective to reduce adverse effects due to rapid altitude change.

The method of claim 1 1 wherein the modulation of the energy metabolism or cytokine production in the mammal is effective to increase memory function.

The method of claim 1 1 wherein the modulation of the energy metabolism or cytokine production in the mammal is effective to alleviate a symptom associated with a respiratory condition.