Compositions and methods to treat or prevent metabolic fatigue using at compound oleuropein or metabolite thereof

Abstract

To provide a method of treating or preventing a mitochondria-related disease or a condition associated with altered mitochondrial function in an individual in need thereof or at risk thereof.SOLUTION: There is provided a method for achieving at least one result selected from the group consisting of (i) improvement in a physiological state linked to metabolic fatigue in one or more cells, (ii) increased mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treatment or prevention of a calcium deficiency / depletion disorder, the method comprises orally administering an effective amount of at least one of oleuropein or metabolite thereof to an individual.SELECTED DRAWING: None

Description

[Background technology]

[0001]

[0001] The present disclosure relates generally to compositions and methods for managing energy at the cellular level using oleuropein or at least one of its metabolites, which can enhance mitochondrial function and increase bioenergetics through activation of the mitochondrial calcium uniporter, thereby promoting cellular activity.

[0002] Sarcopenia is defined as the age-related decline in muscle mass and muscle function (including muscle strength and walking speed). Muscle function and physical performance decline with muscle mass. Impaired muscle function is a highly predictive indicator of bedriddenness, disability, and death in older adults. With the increasing elderly population, the prevalence of sarcopenia is increasing, with 45% of the elderly population in the United States experiencing moderate to severe symptoms. Direct and indirect medical costs attributable to sarcopenia in the United States amount to nearly $19 billion. Therefore, preventing and / or treating sarcopenia could have a significant impact on public health and quality of life, as well as on healthcare economics. Unfortunately, the etiology and physiopathological mechanisms of sarcopenia remain poorly understood, making it difficult to develop effective preventive or therapeutic strategies.

[0003] [Summary of the Invention]

[0003] Mitochondria are the main source of aerobic energy production in mammalian cells and contain large Ca2+ molecules across their inner membranes. 2+ It maintains the Ca gradient and provides the signaling potential for the molecule. 2+ It may function in mitochondria to regulate ATP production and contribute to the orchestration of cellular metabolic homeostasis. (Glancy, B. and RS Balaban (2012). "Role of mitochondrial Ca2+ in the regulation of cellular energetics." Biochemistry 51(14):2959-2973)

[0004]

[0004] In view of the experimental data disclosed later in this specification, the inventors believe that oleuropein enhances mitochondrial efficiency to produce energy, and that optional calcium can enhance this effect from oleuropein.

[0005]

[0005] Thus, in a general embodiment, the present disclosure provides a method for achieving at least one result selected from the group consisting of (i) improving a physiological condition associated with metabolic fatigue in one or more cells, (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treating or preventing a calcium deficiency / depletion disorder, comprising orally administering to an individual an effective amount of oleuropein or at least one of its metabolites.

[0006] In one embodiment, at least a portion of the one or more cells is part of at least one body part selected from the group consisting of liver, kidney, brain, and skeletal muscle.

[0007] In one embodiment, the physiological conditions associated with metabolic fatigue include muscle fatigue or weakness, lack of energy, lack of physical energy, lack of strength or weakness.

[0008] In one embodiment, an effective amount of oleuropein or at least one of its metabolites is administered daily for at least one week.

[0009]

[0009] In one embodiment, the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

[0010]

[0010] In one embodiment, an effective amount of oleuropein or at least one of its metabolites is administered in a composition selected from the group consisting of food compositions, beverages, dietary supplements, nutritional compositions, nutraceuticals, powdered nutritional products to be reconstituted with water or milk before ingestion, food additives, medicines, drinks, pet foods, and combinations thereof.

[0011] In one embodiment, oleuropein or at least one of its metabolites is administered in a composition further comprising calcium.

[0012]

[0012] In one embodiment, an effective amount of oleuropein or at least one of its metabolites is administered in a food product further comprising an ingredient selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0013] In another embodiment, the present disclosure provides a method for treating or preventing (e.g., reducing the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need of or at risk for such treatment, comprising orally administering to the individual an effective amount of oleuropein or at least one of its metabolites.

[0014]

[0014] The mitochondrial-related disease or condition can be selected from the group consisting of stress, physiological aging, obesity, metabolic slowing, metabolic syndrome, diabetes mellitus, complications from diabetes, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy, and dystrophies, and combinations thereof.

[0015] In one embodiment, oleuropein or at least one of its metabolites is administered in a composition further comprising calcium.

[0016]

[0016] In another embodiment, the present disclosure provides a unit dosage form comprising oleuropein or at least one of its metabolites in an effective amount for at least one outcome selected from the group consisting of: (i) improving a physiological condition associated with metabolic fatigue in one or more cells, (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treating or preventing (e.g., reducing the prevalence and / or severity of) calcium deficiency / depletion disorders. At least a portion of the one or more cells may be part of at least one body part selected from the group consisting of the liver, kidney, brain, and musculoskeletal muscle.

[0017] In one embodiment, the physiological conditions associated with metabolic fatigue include muscle fatigue or weakness, lack of energy, lack of physical energy, lack of strength or weakness.

[0018]

[0018] In one embodiment, the unit dosage form essentially comprises oleuropein or at least one of its metabolites.

[0019]

[0019] In one embodiment, the unit dosage form comprises an excipient and at least one of oleuropein or a metabolite thereof.

[0020] In one embodiment, the unit dosage form further comprises calcium. The unit dosage form may essentially comprise calcium and at least one of oleuropein or its metabolites. The unit dosage form may comprise an excipient, calcium, and at least one of oleuropein or its metabolites.

[0021]

[0021] In another embodiment, the present disclosure provides a method for producing a composition for achieving at least one result selected from the group consisting of: (i) improving a physiological condition associated with metabolic fatigue in one or more cells, (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treating or preventing (e.g., reducing the prevalence and / or severity of) calcium deficiency / depletion disorders. The method comprises adding an effective amount of oleuropein or at least one of its metabolites to at least one raw material selected from the group consisting of protein, carbohydrate, and fat. At least a portion of the one or more cells may be part of at least one body part selected from the group consisting of liver, kidney, brain, and skeletal muscle.

[0022] In one embodiment, the method further comprises adding calcium to at least one raw material.

[0023]

[0023] In one embodiment, the method further comprises adding a food additive selected from the group consisting of an acidulant, a thickener, a pH-adjusting buffer or agent, a chelating agent, a colorant, an emulsifier, an excipient, a flavoring, a mineral, an osmotic agent, a pharmaceutically acceptable carrier, a preservative, a stabilizer, a sugar, a sweetener, a modifier, a vitamin, a mineral, and combinations thereof to at least one raw material.

[0024]

[0024] Additional features and advantages are described herein, and will be apparent from the drawings and detailed description that follow. [Brief explanation of the drawings]

[0025]

[0025] [Figure 1] 1 shows the chemical structure of oleuropein. [Figure 2] Based on literature findings, putative pathways for oleuropein metabolism by mammalian and microbial enzymes are shown. [Figure 3A]The chemical structure of homovanillyl alcohol is shown. [Figure 3B] This shows an isomer of homovanillyl alcohol (3-hydroxy-4-methoxyphenethanol or 3-hydroxy-4-methoxyphenethyl alcohol). [Figure 4] Graph showing that oleuropein increases mitochondrial calcium elevation in HeLa cells during stimulation. Statistical evaluation of the effect of oleuropein (10 μM, black) on the integrated mitochondrial calcium elevation elicited by 100 μM histamine. Graph shows the average of three independent experiments. Results are expressed as mean ± SEM. * indicates statistically significant difference vs. control cells (white) at P<0.05 (Student's t-test). [Figure 5] Graph showing that oleuropein enhances mitochondrial calcium in caffeine-stimulated myotubes differentiated from human skeletal muscle myoblasts (HSMM). Statistical evaluation of the effect of oleuropein (10 μM, black) on the integrated mitochondrial calcium rise elicited by 5 mM caffeine. Graph shows the average of six independent experiments. Results are expressed as mean ± SEM. * indicates statistically significant difference vs. control cells (white) at P<0.05 (Student's t-test). [Figure 6] Graph showing that oleuropein metabolites enhance mitochondrial calcium in caffeine-stimulated HSMM myotubes. Statistical evaluation of the effect of oleuropein and its metabolites at a concentration of 10 mM on the integrated mitochondrial calcium increase evoked by 5 mM caffeine. Graph shows the average of six independent experiments. Selected metabolites are on the right. Results are expressed as mean ± SEM. * indicates a statistically significant difference at P<0.05 (one-way ANOVA test) compared to control cells (white). [Figure 7]Graph showing that Ca2+ supplementation enhances mitochondrial Ca2+ elevation in a dose / response manner in C2C12-derived myotubes. Statistical evaluation of the effect of extracellular calcium abundance on the integrated mitochondrial calcium elevation evoked by 5 mM caffeine. On the right is the calcium concentration (mM) in the medium. Graph shows the average of 12 measurements from three independent experiments. Results are expressed as mean ± SEM. * indicates a statistically significant difference at P<0.05 (one-way ANOVA test) compared to a calcium concentration of 0.5 mM in the medium (white). [Figure 8] Graph showing that oleuropein rescues mitochondrial activation under calcium-deficient conditions in C2C12-derived myotubes. Statistical evaluation of the effect of 50 μM oleuropein on the increase in integrated mitochondrial calcium evoked by 5 mM caffeine. Calcium concentration (mM) in the medium is shown on the right. The graph shows the average of 12 measurements from three independent experiments. Results are expressed as mean ± SEM. * indicates a statistically significant difference at P<0.05 (one-way ANOVA test) compared to a calcium concentration of 0.5 mM in the medium (white). [Figure 9] Figure 1 shows graphs demonstrating that oleuropein and hydroxytyrosol enhance the ATP synthase-dependent component of respiration during stimulation in myotubes differentiated from human skeletal muscle (HSM) myoblasts. Statistical evaluation of the effect of 10 μM hydroxytyrosol (gray bars) or 10 μM oleuropein (black bars) on the ATP synthase-dependent component of respiration in HSM myotubes stimulated with 10 μM epibatidine, calculated from the data in the inset. The inset shows the respiratory profile of human skeletal muscle myotubes. The compounds are hydroxytyrosol or oleuropein. Oligomycin was used to determine the ATP synthase-dependent component of respiration in epibatidine-stimulated myotubes. The graph shows the average of eight experiments. Results are expressed as mean ± SEM. * indicates a statistically significant difference vs. control (white bars) at P < 0.05 (one-way ANOVA test). [Figure 10]1 is a graph showing that oleuropein increases ATP production in caffeine-stimulated C2C12-derived myotubes. Myotubes were incubated with oleuropein for 15 minutes and then stimulated with 5 mM caffeine for 10 minutes. The graph shows the average of eight experiments. Results are expressed as mean ± SEM. * indicates a statistically significant difference vs. control cells (white) at P<0.05 (Student's t-test). [Figure 11] Figure 1 shows graphs demonstrating that oleuropein increases mitochondrial calcium uptake (ex vivo) in isolated adult mouse muscle fibers transfected with the mitochondrial calcium sensor 4mtGCaMP6f. Fibers were treated with oleuropein. After 3 minutes, cells were stimulated with 60 mM caffeine. Left: Representative trace of mitochondrial calcium uptake. Right: Mean of mitochondrial calcium peaks. Results are expressed as mean ± SD. * indicates a statistically significant difference vs. control muscle fibers at P<0.05 (Student's t-test) for >20 muscle fibers per condition. [Figure 12] Figure 1 shows graphs demonstrating that hydroxytyrosol increases mitochondrial calcium uptake in isolated adult mouse myofibers transfected with the mitochondrial calcium sensor 4mtGCaMP6f (ex vivo). Fibers were treated with hydroxytyrosol. After 3 minutes, cells were stimulated with 60 mM caffeine. Left: Representative trace of mitochondrial calcium uptake. Right: Mean of mitochondrial calcium peaks. Results are expressed as mean ± SD. * indicates a statistically significant difference vs. control myofibers at P<0.05 (Student's t-test) for >20 myofibers per condition. [Figure 13]This graph shows that oleuropein increases mitochondrial respiration in isolated adult mouse muscle fibers (ex vivo). Fibers treated with oleuropein for 2 hours were placed in an XF24 extracellular flux analyzer (Agilent) to measure the caffeine-stimulated oxygen consumption rate. Oligomycin, FCCP, and antimycin / rotenone were added sequentially to calculate basal, maximal, ATP-related, and non-mitochondrial respiration. Results are expressed as mean ± SD. * indicates a statistically significant difference from control muscle fibers at P < 0.05 (Student's t-test) for 7 wells per condition. [Figure 14] This graph shows that hydroxytyrosol increases mitochondrial respiration in isolated adult mouse muscle fibers (ex vivo). Fibers treated with hydroxytyrosol for 2 hours were placed in an XF24 extracellular flux analyzer (Agilent) to measure the caffeine-stimulated oxygen consumption rate. Oligomycin, FCCP, and antimycin / rotenone were added sequentially to calculate basal, maximal, ATP-related, and non-mitochondrial respiration. Results are expressed as mean ± SD. * indicates a statistically significant difference from control muscle fibers at P < 0.05 (Student's t-test) for 7 wells per condition. [Figure 15] Figure 1 is a graph showing that oleuropein increases resistance to fatigue in mouse EDL (extensor digitorum longus) muscles (ex vivo). Muscles incubated with oleuropein exhibit a significantly slower muscle weakness upon fatigue than muscles treated with DMSO. The third, fourth, and fifth tetanic stimulations were significantly higher for oleuropein compared to the control, suggesting a higher resistance to fatigue. P values are indicated for each tetanic contraction. Results are expressed as mean ± SD. * indicates a statistically significant difference vs. control muscles at P<0.05 (Student's t-test). Each experiment was repeated in 10 muscles for both experimental groups. [Figure 16]Figure 1 shows that Bonolive® (BioActor BV, NL), an olive leaf extract standardized for oleuropein content (≥ 40% oleuropein and < 1% hydroxytyrosol (OHT)), promotes mitochondrial activation by dephosphorylation of pyruvate dehydrogenase (PDH) in aged rats treated for 3 months. Phospho-PDH and PDH levels were analyzed in gastrocnemius muscle in control and 3-month treated animals (insert). Mitochondrial PDH activation was measured as the ratio of total PDH to phospho-PDH levels. The graph shows the average of muscles from 5 animals. Results are expressed as mean ± SEM. * indicates statistically significant difference vs. control (white bar) at P < 0.05 (Student's t-test). DETAILED DESCRIPTION OF THE INVENTION

[0026]

[0041] definition

[0042] Some definitions are provided below. However, definitions may be found in the "Embodiments" section below, and the heading "Definitions" above does not imply that such disclosure in the "Embodiments" section is not a definition.

[0027]

[0043] All percentages set forth herein are by weight of the total composition unless otherwise specified. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers within a numerical range, e.g., within -10% to +10% of the referenced number, preferably within -5% to +5% of the referenced number, more preferably within -1% to +1% of the referenced number, and most preferably within -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers or fractions within that range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within that range. For example, a disclosure of 1 to 10 should be interpreted as supporting ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0028]

[0044] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural references unless otherwise indicated. Thus, for example, reference to "a metabolite" or "the metabolite" includes one metabolite, but also includes two or more metabolites.

[0029]

[0045] The terms "comprise," "comprises," and "comprising" should be construed as inclusive rather than exclusive. Similarly, the terms "include," "including," and "or" should all be construed as inclusive unless such a construction is clearly prevented by the context. However, the compositions disclosed herein may not include elements not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments "consisting essentially of," as well as embodiments "comprising of," the specified components.

[0030]

[0046] As used herein, "a composition essentially comprising oleuropein or at least one metabolite thereof" and "a composition essentially comprising calcium and oleuropein or at least one metabolite thereof" do not include any additional compounds that affect mitochondrial calcium transport other than oleuropein or at least one metabolite thereof and optional calcium. In certain non-limiting embodiments, the composition consists of an excipient, oleuropein or at least one metabolite thereof, and optionally calcium.

[0031]

[0047] The term "and / or" used in the context of "X and / or Y" should be interpreted as "X" or "Y" or "X and Y." Similarly, "at least one of X or Y" should be interpreted as "X" or "Y" or "both X and Y." For example, "at least one of oleuropein or a metabolite thereof" means "oleuropein" or "a metabolite of oleuropein" or "both oleuropein and a metabolite thereof."

[0032]

[0048] As used herein, the terms "example" and "such as," particularly when followed by a list of terms, are merely exemplary and illustrative and should not be considered exclusive or exhaustive. As used herein, "associated with" and "linked with" mean occurring simultaneously, preferably caused by the same underlying condition, and most preferably, one of the specified conditions is caused by the other specified condition.

[0033]

[0049] The terms "food," "food product," and "food composition" refer to a product or composition intended for consumption by an individual, such as a human, and which provides at least one nutrient to such an individual. The compositions of the present disclosure, including the many embodiments described herein, can comprise, consist of, or consist essentially of the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described or not described herein that are useful in a diet.

[0034]

[0050] As used herein, the terms "treat" and "treatment" refer to administering a composition disclosed herein to a subject having a condition with the intent of attenuating, reducing, or ameliorating at least one symptom associated with the condition and / or slowing, reducing, or preventing the progression of the condition. The terms "treatment / therapy" and "treating" include both preventative or prophylactic treatments (treatments that prevent and / or slow the onset or progression of a targeted pathological condition or disorder) and curative, therapeutic, or disease-modifying treatments, including, for example, therapeutic measures to cure, delay, alleviate symptoms, and / or halt the progression of a diagnosed pathological condition or disorder, as well as treatment of patients at risk of or suspected of having the condition, and patients who are unwell or diagnosed with a disease or medical condition. The terms "treatment / therapy" and "treating" do not necessarily imply treating the subject until full recovery. The terms "treatment" and "treating" also refer to maintaining and / or promoting the health of an individual who is not afflicted with a disease but who may be susceptible to an ill-health condition. The terms "treatment" and "treating" are also intended to include the synergism or otherwise enhancement of one or more primary preventative or therapeutic measures. By way of non-limiting example, treatment may be performed by the patient, a caregiver, a doctor, a nurse, or another medical professional.

[0035]

[0051] Both human and animal treatments are within the scope of this disclosure. Preferably, oleuropein or at least one of its metabolites is administered in a serving or unit dosage form that provides a therapeutically or prophylactically effective amount.

[0036]

[0052] The terms "prevent" and "prevention" refer to administering a composition disclosed herein to a subject not exhibiting any symptoms of the condition to reduce or prevent the onset of at least one symptom associated with the condition. Furthermore, "prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder.

[0037]

[0053] As used herein, an "effective amount" is an amount that treats or prevents a deficiency, treats or prevents a disease or medical condition in an individual, or, more generally, alleviates symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to an individual.

[0038]

[0054] The relative terms "improved," "increased / enhanced / boosted," "potentiated," and the like refer to the effect of an effective amount of a composition disclosed herein, i.e., a composition comprising oleuropein or at least one of its metabolites, compared to the administration of an identical composition, but lacking oleuropein and oleuropein metabolites, over the same period of time.

[0039]

[0055] As used herein, "administering" includes another individual providing the referenced composition to an individual so that the individual can ingest the composition, and also simply includes the individual's own act of ingesting the referenced composition.

[0040]

[0056] "Animal" includes mammals, including, but not limited to, rodents, aquatic mammals, domestic animals such as dogs, cats, and other pets, livestock such as sheep, pigs, cattle, and horses, and humans. When "animal," "mammal," or their plural forms are used, these terms also apply to any animal in which the effect exhibited or intended by the context of the passage can be exhibited, e.g., the animal benefiting from improved mitochondrial calcium transport. The term "individual" or "subject" is often used herein to refer to humans, although the disclosure is not so limited. Thus, the term "individual" or "subject" refers to any animal, mammal, or human that can benefit from the methods and compositions disclosed herein.

[0041]

[0057] The term "pet" refers to any animal that can benefit from or enjoy the compositions provided by the present disclosure. For example, a pet may be an animal such as a bird, bovine, canine, equine, feline, caprine, wolf, murine, ovine, or porcine, although a pet may be any suitable animal. The term "companion animal" refers to a dog or cat.

[0042]

[0058] The term "elderly," in relation to humans, refers to an age of at least 60 years, preferably greater than 63 years, more preferably greater than 65 years, and most preferably greater than 70 years. In the context of non-human animals, "elderly" refers to a non-human subject that is likely to have reached 60% of its lifespan, and in some embodiments, at least 70%, at least 80%, or at least 90% of its lifespan. Determination of lifespan may be based on actuarial tables, calculations, or estimates and may take into account past, present, and future influences or factors known to positively or negatively affect lifespan. When determining lifespan, species, sex, body size, genetic factors, environmental factors and stressors, current and past health status, past and present nutritional status, and stressors may be considered. The term "older adult" in the human context refers to a postnatal age of 45 years or older, preferably greater than 50 years, and more preferably greater than 55 years, and includes elderly individuals.

[0043]

[0059] The terms "serving" and "unit dosage form," as used herein, are interchangeable and refer to physically discrete units suitable as unitary doses for human and animal subjects, each unit containing a predetermined amount of a composition comprising oleuropein or at least one of its metabolites, as disclosed herein, preferably with a pharmaceutically acceptable diluent, carrier, or vehicle, in an amount sufficient to produce a desired effect. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In one embodiment, the unit dosage form may be a predetermined amount of liquid contained in a container such as a bottle.

[0044]

[0060] An "oral nutritional supplement" or "ONS" is a composition comprising at least one macronutrient and / or at least one micronutrient, e.g., in a sterile liquid, semi-solid, or powder form, intended to supplement other nutritional intakes, such as those from food. Non-limiting examples of commercially available ONS products include MERITENE®, BOOST®, NUTREN®, and SUSTAGEN®. In some embodiments, an ONS can be a liquid beverage that can be consumed without the addition of additional liquid, e.g., the liquid volume is one serving of the composition.

[0045]

[0061] As used herein, "incomplete nutrition" preferably refers to a nutritional product that does not contain sufficient levels of macronutrients (protein, fat, and carbohydrates) or micronutrients to be the sole source of nutrition for the animal to which it is administered. The term "complete nutrition" refers to a product that can be the sole source of nutrition for a subject. An individual can obtain 100% of their nutritional needs from a complete nutritional composition.

[0046]

[0062] The term "kit" means that the components of the kit in or with one or more containers are physically associated and considered as a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, packaging of any type, design, or material, overwrap, shrink wrap, affixed components (e.g., stapled or glued), or packaging of combinations thereof.

[0047]

[0063] "Metabolic fatigue" refers to a decrease in mitochondrial function in one or more cells (e.g., one or more of the liver, kidney, brain, and skeletal muscle) due to a lack of substrates within one or more cells and / or accumulation of metabolic products within muscle fibers, which inhibits either calcium release or the ability of calcium to stimulate mitochondrial function. Physiological conditions associated with metabolic fatigue can include muscle fatigue or weakness, lack of energy, particularly lack of physical energy, lack of strength, or weakness.

[0048]

[0064] Embodiment

[0065] Oleuropein is a polyphenol found in the fruits, roots, stems, and more specifically, leaves of plants belonging to the Oleaceae family, particularly olives (Olea europaea). Figure 1 shows the chemical structure of oleuropein. Oleuropein is a heterosidic ester of 3,4-dihydroxyphenylethanol (also known as hydroxytyrosol, labeled "A" in Figure 1) and elenolic acid (labeled "B" in Figure 1) containing a glucose molecule (labeled "C" in Figure 1). Figure 2 shows the proposed metabolic pathway of oleuropein by mammalian and microbial enzymes, based on findings reported in the literature.

[0049]

[0066] Another aspect of the present disclosure is a method for achieving at least one result selected from the group consisting of (i) improving a physiological condition associated with metabolic fatigue in one or more cells, (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treating or preventing (e.g., reducing the prevalence and / or severity of) calcium deficiency / depletion disorders, comprising orally administering to an individual an effective amount of oleuropein or at least one of its metabolites.

[0050]

[0067] Another aspect of the present disclosure is a method for treating at least one condition selected from the group consisting of (i) physiological conditions associated with metabolic fatigue in one or more cells and (ii) calcium deficiency / depletion disorders in an individual in need thereof or preventing at risk thereof (e.g., reducing the incidence and / or severity), the method comprising orally administering to an individual in need thereof or at risk thereof an effective amount of oleuropein or at least one of its metabolites.

[0051]

[0068] Yet another aspect of the present disclosure is a method for treating or preventing (e.g., reducing the incidence and / or severity of) a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need of such treatment or at risk thereof. The method comprises orally administering to the individual an effective amount of oleuropein or at least one of its metabolites. The mitochondrial-associated disease or condition can be selected from the group consisting of stress, physiological aging, obesity, metabolic slowing, metabolic syndrome, diabetes mellitus, complications from diabetes, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorder, anxiety disorder, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy, and dystrophy, and combinations thereof.

[0052]

[0069] In another embodiment, metabolic fatigue includes lack of energy, particularly physical energy, lack of stamina or weakness.

[0053]

[0070] In some embodiments, the methods include identifying the individual as having or at risk for the condition prior to administration.

[0054]

[0071] The effective amount of oleuropein or at least one metabolite thereof will vary depending on the particular composition, the age and condition of the recipient, and the particular disorder or disease being treated. Nevertheless, in general embodiments, an individual can be administered 0.001 mg to 1.0 g of oleuropein or at least one metabolite thereof per day, preferably 0.01 mg to 0.9 g of oleuropein or at least one metabolite thereof per day, more preferably 0.1 mg to 750 mg of oleuropein or at least one metabolite thereof per day, more preferably 0.5 mg to 500 mg of oleuropein or at least one metabolite thereof per day, and most preferably 1.0 mg to 200 mg of oleuropein or at least one metabolite thereof per day.

[0055]

[0072] In some embodiments, oleuropein is administered in a composition further comprising calcium, at least a portion of which may be one or more calcium salts, such as calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluconate, calcium lactate, or mixtures thereof. In general embodiments, an individual is administered between 0.1 g and 1.0 g of calcium per day, preferably between 125 mg and 950 mg of calcium per day, more preferably between 150 mg and 900 mg of calcium per day, more preferably between 175 mg and 850 mg of calcium per day, and most preferably between 200 mg and 800 mg of calcium per day.

[0056]

[0073] In one embodiment, at least a portion of the oleuropein is obtained by extraction, for example, from one or more of the stems, leaves, fruits, or pits of a plant belonging to the Oleaceae family, preferably a plant belonging to the Oleaceae family, such as olive (olive tree), Ligustrum, Syringa, Fraximus, Jasminum, Osmanthus, etc. Additionally or alternatively, at least a portion of the oleuropein and / or its metabolites can be obtained by chemical synthesis.

[0057]

[0074] Non-limiting examples of suitable metabolites of oleuropein include oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, their glucuronidated forms, their sulfated forms, their derivatives, and mixtures thereof. Figure 3A shows the chemical structure of homovanillyl alcohol. Figure 3B shows its isomer (3-hydroxy-4-methoxyphenethanol or 3-hydroxy-4-methoxyphenethyl alcohol).

[0058]

[0075] In some embodiments, oleuropein or at least one of its metabolites is the only polyphenol in the composition and / or the only polyphenol administered to the individual.

[0059]

[0076] In some embodiments, oleuropein or at least one of its metabolites, and optional calcium, can be administered to an elderly subject. In some embodiments, the individual is healthy. In some embodiments, the individual has metabolic fatigue, but optionally is otherwise healthy. In other embodiments, the individual may be a pet.

[0060]

[0077] In one embodiment, at least a portion of the one or more cells are part of at least one body part selected from the group consisting of liver, kidney, brain, and skeletal muscle.

[0061]

[0078] Oleuropein or at least one of its metabolites, and optional calcium can be administered in any composition suitable for human and / or animal consumption.In a preferred embodiment, oleuropein or at least one of its metabolites, and optional calcium are administered to individuals orally or enterally (e.g., tube feeding).For example, oleuropein or at least one of its metabolites, and optional calcium can be administered to individuals in a drink, food product, capsule, tablet, powder, or suspension.

[0062]

[0079] Non-limiting examples of suitable compositions include food compositions, dietary supplements, nutritional supplements (e.g., liquid ONS), complete nutritional compositions, beverages, pharmaceuticals, nutraceuticals, powdered nutritional products to be reconstituted with water or milk before consumption, food additives, medicines, drinks, pet foods, and combinations thereof.

[0063]

[0080] Food products according to the present invention may include fermented dairy products, such as yogurt, buttermilk, and the like; ice cream; condensed milk; milk; dairy cream; flavored milk drinks; whey-based drinks; toppings; coffee creamers; chocolate; cheese-based products; soups; sauces; purees; dressings; puddings; custards; infant foods; nutritional formulas, such as complete nutritional formulas for infants, children, teenagers, adults, the elderly, or the seriously ill; and cereals and cereal bars.

[0064]

[0081] Drinks may include, for example, milk or yogurt-based drinks, fermented milks, protein drinks, coffee, tea, energy drinks, soy drinks, fruit and / or vegetable drinks, and fruit and / or vegetable juices.

[0065]

[0082] The oleuropein or at least one of its metabolites, and optional calcium, may be administered in a food product further comprising an ingredient selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0066]

[0083] When oral or enteral administration is not possible or advisable, the compositions can be administered parenterally.

[0067]

[0084] In another embodiment, the present disclosure provides a method for treating or preventing (e.g., reducing the incidence and / or severity of) a mitochondrial-associated disease or condition associated with altered mitochondrial function in an individual in need of or at risk for such treatment, comprising orally administering to the individual in need of or at risk for such treatment an effective amount of oleuropein or at least one of its metabolites.

[0068]

[0085] In one embodiment, oleuropein or at least one of its metabolites and optional calcium are administered to an individual for a period of at least one month, preferably at least two months, more preferably at least three, four, five, or six months, and most preferably at least one year. During this period, oleuropein or at least one of its metabolites and optional calcium can be administered to an individual at least one day per week, preferably at least two days per week, more preferably at least three, four, five, or six days per week, and most preferably seven days per week. Oleuropein or at least one of its metabolites and optional calcium can be administered in a single dose per day or in multiple separate doses per day.

[0069]

[0086] The above dosing examples do not require uninterrupted daily administration. Rather, administration may be interrupted by several short periods, such as a 2-4 day break during the administration period. The ideal duration of administration of the present compositions can be determined by one of skill in the art.

[0070]

[0087] In one embodiment, oleuropein or at least one of its metabolites can be administered together with calcium in the same composition, for example, in a unit dosage form containing both calcium and oleuropein or at least one of its metabolites.

[0071]

[0088] In an alternative embodiment, oleuropein or at least one of its metabolites can be administered sequentially in separate compositions. The term "sequentially" refers to sequential administration of calcium and oleuropein or at least one of its metabolites, such that oleuropein or at least one of its metabolites is administered at a first time without calcium, and calcium is administered at a second time (before or after the first time) without oleuropein or at least one of its metabolites. The time between consecutive administrations can be, for example, one or several seconds, minutes, or hours on the same day; one or several days, or several weeks on the same month; or one or several months on the same year.

[0072]

[0089] Another aspect of the present disclosure is a method for producing a composition to achieve an effect selected from the group consisting of: (i) improving a physiological condition associated with metabolic fatigue in one or more cells; (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells; and (iii) treating or preventing (e.g., reducing the prevalence and / or severity of) calcium deficiency / depletion disorders.

[0073]

[0090] The method includes adding at least one of oleuropein or its metabolites to an ingredient selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof. The composition (e.g., a food product) can be made prior to administration (e.g., the composition is made, packaged, and then purchased by a consumer who administers the composition to themselves or another individual) or can be made substantially simultaneously with administration (the composition is made by an individual who administers the composition to themselves or another individual less than 30 minutes prior to administration, preferably less than 15 minutes prior to administration, more preferably less than 10 minutes prior to administration, and most preferably less than 5 minutes prior to administration).

[0074]

[0091] The composition may comprise an effective amount of oleuropein or at least one of its metabolites. For example, a single serving or dose of the composition may contain an effective amount, and a package may contain one or more servings or doses. Optionally, the composition may further comprise calcium.

[0075]

[0092] The composition may include a food additive selected from the group consisting of an acidulant, a thickener, a pH-adjusting buffer or agent, a chelating agent, a colorant, an emulsifier, an excipient, a flavoring agent, a mineral, an osmotic agent, a pharmaceutically acceptable carrier, a preservative, a stabilizer, a sugar, a sweetener, a modifier, a vitamin, a mineral, and combinations thereof.

[0076]

[0093] In addition to oleuropein or at least one of its metabolites and optional calcium, the composition can further comprise an animal- or plant-derived protein source, such as milk protein, soy protein, and / or pea protein. In a preferred embodiment, the protein source is selected from the group consisting of whey protein, casein protein, pea protein, soy protein, wheat protein, corn protein, rice protein, proteins derived from legumes, cereals, and grains, and combinations thereof. Additionally or alternatively, the protein source can comprise proteins derived from nuts and / or seeds.

[0077]

[0094] The protein source preferably comprises whey protein. The whey protein may be hydrolyzed or non-hydrolyzed. The whey protein may be any whey protein, for example, the whey protein may be selected from the group consisting of whey protein concentrate, whey protein isolate, whey protein micelles, whey protein hydrolysate, acid whey, sweet whey, denatured sweet whey (sweet whey from which caseinoglycomacropeptide has been removed), whey protein fractions, and any combination thereof. In a preferred embodiment, the whey protein comprises whey protein isolate and / or denatured sweet whey.

[0078]

[0095] As mentioned above, the protein source may be of animal or plant origin, for example, milk protein, soy protein, and / or pea protein. In one embodiment, the protein source comprises casein. Casein can be obtained from any mammal, but is preferably obtained from cow's milk, and preferably as casein micelles.

[0079]

[0096] The composition may include one or more branched-chain amino acids. For example, the composition may include leucine, isoleucine, and / or valine. The protein source of the composition may include leucine in its free form and / or leucine bound as a peptide and / or protein, such as dairy protein, animal protein, or vegetable protein. In one embodiment, the composition includes leucine in an amount of up to 10% by weight of the dry matter of the composition. Leucine may be present as D-leucine or L-leucine, preferably in the L-form. When the composition includes leucine, the composition may be administered in a daily dose providing 0.01-0.04 g of leucine per kg of body weight, preferably 0.02-0.035 g of leucine per kg of body weight. While such doses are particularly appropriate for complete nutritional compositions, those skilled in the art will readily recognize how to adjust the dose for oral nutritional supplements (ONS).

[0080]

[0097] In addition to any calcium, one or more other minerals can be used in the composition. Non-limiting examples of suitable minerals include boron, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.

[0081]

[0098] One or more additional vitamins can be used in the composition. Non-limiting examples of suitable vitamins include vitamin A, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin or niacinamide), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine, pyridoxal, pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; commonly cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, folic acid, and biotin), and combinations thereof. "Vitamins" include compounds such as provitamins, their derivatives, and their analogs, which are naturally obtained from plant and animal foods or synthesized.

[0082]

[0099] The composition may also contain a carbohydrate source and / or a fat source. Non-limiting examples of suitable fats include canola oil, corn oil, and high oleic sunflower oil. Non-limiting examples of suitable carbohydrates include sucrose, lactose, glucose, fructose, corn syrup solids, maltodextrin, and mixtures thereof. Additionally or alternatively, dietary fiber may be added. Dietary fiber passes through the small intestine without being enzymatically digested and functions as a natural bulking agent and laxative. Dietary fiber may be soluble or insoluble, with a blend of the two generally being preferred. Non-limiting examples of suitable dietary fiber include soybean, pea, oat, pectin, guar gum, partially hydrolyzed guar gum, gum arabic, fructooligosaccharides, acid oligosaccharides, galactooligosaccharides, sialyllactose, and oligosaccharides derived from animal milk. A preferred fiber blend is a mixture of inulin and relatively short-chain fructooligosaccharides. In one embodiment, the fiber content is from 2 to 40 g / L of the composition, for example from 4 to 10 g / L.

[0083]

[0100] One or more food-grade emulsifiers can be incorporated into the composition, such as diacetyl tartaric acid esters of mono- and diglycerides, lecithin, and / or mono- and diglycerides. Suitable salts and stabilizers can be included.

[0101] [Example]

[0084]

[0102] The following non-limiting examples provide experimental data supporting the compositions and methods disclosed herein.

[0085]

[0103] Example 1

[0104] To test the effects of oleuropein, its metabolites, and calcium supplementation / depletion in living cells, we measured mitochondrial calcium elevation in HeLa cells and myotubes differentiated from both mouse C2C12 cells and human primary adult muscle cells. HeLa and C2C12 cells were purchased from ATCC. Human skeletal muscle myoblasts (HSSM) were purchased from Lonza. HSMMs were isolated from the arm or leg muscle tissue of healthy donors and used after the second passage. HeLa cells were seeded in 96-well plates at a density of 50,000 cells / well in minimal essential medium (DMEM, Gibco), high glucose, plus 10% fetal bovine serum. C2C12 cells were seeded in 96-well plates at a density of 8,000 cells / well in DMEM high glucose (Gibco) plus 10% fetal bovine serum. Myotubes were differentiated from C2C12 cells by growing the cells for 4 days in DMEM containing 2% horse serum. HSMM were seeded in 96-well plates at a density of 8000 cells / well in DMEM / F-12 (Gibco). Myotubes were differentiated from HSMM by growing the cells for 4 days in SKM-M (ZenBio) containing 2% horse serum.

[0086]

[0105] Mitochondrial calcium measurements were performed using HeLa cells or myotubes (Sirion Biotech) infected with an adenovirus expressing the mitochondrial-targeted calcium sensor mitochondrial modified aequorin (Montero et al., 2004). For aequorin reconstitution, 24 h after infection, cells or myotubes were incubated with 1 μM wild-type coelenterazine in standard medium (145 mM NaCl, 5 mM KCl, 1 mM MgCl, 1 mM CaCl, 10 mM glucose, and 10 mM Hepes) for 2 h at room temperature (22°C).

[0087]

[0106] For treatment, compounds were added directly to cell or myotube cultures 2 hours before measurement. Luminescence was measured using a Cytation 3 cell imaging reader (Bioteck) or a FLIPR Tetra Aequorin (Molecular Devixes). Conversion of luminescence data to calcium concentrations (calibration) was performed using an algorithm previously described (Alvarez & Montero, 2002). Quantification was performed using a custom module analysis based on Excel (Microsoft) and GhaphPad Prism 7.02 (GhaphPad) software.

[0088]

[0107] As shown in Figure 4, oleuropein increases mitochondrial calcium elevation in HeLa cells during stimulation. As shown in Figure 5, oleuropein activates mitochondrial calcium in caffeine-stimulated myotubes differentiated from human skeletal muscle myoblasts (HSMM). As shown in Figure 6, phenolic metabolites of oleuropein activate mitochondrial calcium in caffeine-stimulated HSMM myotubes. As shown in Figure 7, Ca 2+ Supplementation of ATP upregulates mitochondrial Ca in a dose / response manner in C2C12-derived myotubes. 2+ As shown in Figure 8, oleuropein rescues mitochondrial activation under conditions of calcium depletion in C2C12-derived myotubes.

[0089]

[0108] Example 2

[0109] To examine the effects of oleuropein and hydroxytyrosol on mitochondrial respiration and evaluate the effects of these compounds on the ATP synthase-dependent component of respiration, we measured oxygen consumption in human skeletal muscle myotubes. For respiration experiments, oxygen consumption was measured in myotubes using an XF96 instrument (Seahorse Biosciences, MA). Human myotubes were seeded on polyornithine-coated Seahorse tissue plates on day 2 and thereafter, and the cells were washed twice with Krebs-Ringer bicarbonate-HEPES buffer (KRBH) at pH 7.4, containing 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM HEPES, 5 mM NaHCO3, and 10 mM glucose. Respiration rates were measured every 6 minutes at 37°C. ATP synthase-dependent respiration was calculated as the difference in respiration rate before and after addition of oligomycin. Experiments were performed at 37°C.

[0090]

[0110] As shown in Figure 9, oleuropein and hydroxytyrosol enhance ATP synthase-dependent components related to respiration in human skeletal myotubes during stimulation.

[0091]

[0111] Example 3

[0112] To test the effect of oleuropein on ATP production, we measured ATP in myotubes differentiated from C2C12 cells. ATP was measured using a conventional luciferin / luciferase method. Myotubes were incubated in KRBH medium and oleuropein was added for 15 minutes. Myotubes were then stimulated with 5 mM caffeine for an additional 10 minutes. Finally, myotubes were incubated with luciferin / luciferase in lysis buffer, and the bioluminescence signal, which is proportional to the amount of ATP present, was measured using a Cytation3 cell imaging reader (Bioteck). As shown in Figure 10, oleuropein increased ATP production in caffeine-stimulated C2C12-derived myotubes.

[0092]

[0113] Example 4

[0114] To test the effects of oleuropein and hydroxytyrosol on mitochondrial calcium uptake in isolated adult mouse muscle fibers, flexor digitorum brevis (FDB) fibers were isolated 7–10 days after in vivo transfection. Muscles were digested with collagenase A (4 mg / mL) (Roche) dissolved in Tyrode's solution (pH 7.4) (Sigma-Aldrich) containing 10% fetal bovine serum (Thermo Fisher Scientific). Single fibers were isolated, plated on laminin-coated glass coverslips, and cultured in DMEM containing HEPES (42430 Thermo Fisher Scientific) supplemented with 10% fetal bovine serum containing penicillin (100 U / mL) and streptomycin (100 μg / mL). Fibers were maintained in culture at 37°C with 5% CO2. Mitochondrial Ca was measured. 2+For measurements, FDB muscles were electroporated with a plasmid encoding the mitochondrial calcium sensor 4mtGCaMP6f. Real-time imaging was performed after single fiber isolation. To avoid fiber contraction during the experiment, muscle fibers were maintained at room temperature in Krebs-Ringer modified buffer (135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES, 1 mM MgSO4, 0.4 mM KH2PO4, 1 mM CaCl2, 5.5 mM glucose, pH 7.4) in the presence of 75 μM N-benzyl-p-toluenesulfonamide (BTS, Sigma-Aldrich). Addition of 60 mM caffeine (Sigma-Aldrich) was shown to trigger calcium release from intracellular stores. Experiments were performed using a Zeiss Axiovert 200 microscope equipped with a 40× / 1.3NA PlanFluor objective. Excitation was performed with a DeltaRAM V high-speed monochromator (Photon Technology International) equipped with a 75W xenon arc lamp. Images were captured with a highly sensitive Evolve512Delta EMCCD (Photometrics). The system was controlled by MetaMorph 7.5 (Molecular Devices) and assembled by Crisel Instruments. Alternatively, the 4mtGCaMP6f sensor was excited at 410 nm and 475 nm per second, respectively, and images were acquired through dual-band emission filters (520 / 40 and 630 / 60) (Chroma). The exposure time was set to 50 ms. Acquisition was performed with an EM gain of 200 and binning of 1. Image analysis was performed using the Fiji distribution in ImageJ software. Background subtraction was performed on images. As shown in Figure 11, oleuropein increases mitochondrial calcium uptake in isolated adult mouse myofibers. As shown in Figure 12, oleuropein increases mitochondrial calcium uptake in isolated adult mouse myofibers.

[0093]

[0115] Example 5

[0116] To test the effects of oleuropein and hydroxytyrosol on mitochondrial respiration in isolated adult mouse muscle fibers, flexor digitorum brevis (FDB) muscles were isolated as follows. Muscles were digested with collagenase A (4 mg / mL) (Roche) dissolved in Tyrode's salt solution (pH 7.4) (Sigma-Aldrich) containing 10% fetal bovine serum (Thermo Fisher Scientific). Single fibers were isolated, plated onto laminin-coated XF24 microplate wells, and cultured in DMEM (D5030 Sigma-Aldrich) supplemented with 1 mM sodium pyruvate, 5 mM glucose, 33 mM NaCl, 15 mM phenol red, 25 mM HEPES, and 1 mM L-Glu in the presence of 75 μM N-benzyl-p-toluenesulfonamide (BTS, Sigma-Aldrich). Fibers were maintained in culture at 37°C under 5% CO for 2 hours. Oxygen consumption rate was assessed in real time using an XF24 extracellular flux analyzer (Agilent). This allows for measurement of changes in oxygen consumption rate (OCR) after up to four sequential additions of compound. A titration with uncoupled FCCP was performed to obtain the FCCP concentration (0.6 μM) that maximally increased OCR. To measure the effects of oleuropein or hydroxytyrosol on stimulated respiration, fibers were stimulated with 10 mM caffeine. Oligomycin (2 μM) was added to assess the ATP synthase-dependent component of respiration. Results were normalized to the fluorescence of calcein (Sigma-Aldrich). Fibers were placed in 2 μM calcein for 30 min. Measurements were performed using a Perkin-Elmer EnVision plate reader in well-scan mode, using a 480 / 20 nm filter for excitation and a 535 / 20 nm filter for emission. As shown in Figure 13, oleuropein increases stimulated mitochondrial respiration and the ATP synthase-dependent component of respiration in isolated adult mouse muscle fibers. As shown in Figure 14, hydroxytyrosol increases stimulated mitochondrial respiration and ATP synthase-dependent respiration in isolated adult mouse muscle fibers.

[0094]

[0117] Example 6

[0118] To test the effect of oleuropein on muscle fatigue in healthy adult mice, extensor digitorum longus muscles were dissected tendon-to-tendon under a stereomicroscope and mounted between force transducers (KG Scientific Instruments, Heidelberg, Germany) in a small chamber, where oxygenated Krebs solution was continuously circulated and maintained at 25°C. Stimulation conditions were optimized, and muscle length was increased until force development was maximized during 90 Hz stimulation. After the first force-frequency measurement, oleuropein was added to the medium at a final concentration of 10 μM. Force-frequency measurements were then taken every 10 min for up to 1 h after addition. After 1 h, a fatigue protocol was applied, including 120 tetanic contractions (100 Hz) of 300 ms duration repeated every second. Fatigue was measured as the force drop relative to the initial force. Each experiment was repeated on 10 muscles for both experimental groups. As shown in Figure 15, muscles incubated with oleuropein exhibited a significantly slower force drop upon fatigue than control muscles, indicating increased resistance to fatigue.

[0095]

[0119] Example 7

[0120] To test the effect of olive leaf extract standardized for oleuropein content (≥40% oleuropein) in activating mitochondria by dephosphorylating pyruvate dehydrogenase (PDH), gastrocnemius muscles were analyzed by Western blotting after 3 months of supplementation with Bonolive® (BioActor BV, NL) from 20-month-old rats. To monitor protein levels, frozen muscles were pulverized using a Qiagen Tissue Lyser, and protein extracts were prepared in an appropriate buffer containing myolysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 10% glycerol, 2% SDS, 1% Triton X-100, Complete EDTA-free protease inhibitor mix (Roche), 1 mM PMSF, 1 mM NaVO3, 5 mM NaF, and 3 mM β-glycerophosphate). 40 μg of total protein was loaded according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis on a commercially available 4-12% acrylamide gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (Thermo Fisher Scientific) by wet electrophoretic transfer. Blots were blocked with 5% nonfat dry milk (Bio-Rad) in TBS-tween (0.5 M Tris, 1.5 M NaCl, 0.01% Tween) for 1 h at room temperature and then incubated with primary antibodies at 4°C. Secondary antibodies were incubated for 1 h at room temperature. The following antibodies were used: anti-phospho-PDH (1:5000, Abcam) and anti-PDH (1:1000, Cell Signaling). The secondary HRP-conjugated antibody was purchased from Bio-Rad and used at a 1:5000 dilution. Mitochondrial PDH activation was measured as the ratio of total PDH to phospho-PDH levels. As shown in Figure 16, Bonolive® (BioActor BV,NL) promotes mitochondrial activation through dephosphorylation of pyruvate dehydrogenase in aged rats supplemented with Bonolive® (BioActor BV,NL) for 3 months.

[0096]

[0121] References

[0122] Alvarez, J., & Montero, M. (2002). Measuring[Ca2+] in the endoplasmic reticulum whith aequorin. Cell Calcium, 32(5-6), 251-260.

[0123] Montero, M., Lobaton, CD, Hernandez-Sanmiguel, E., Santodomingo, J., Vay, L., Moreno, A., & Alvarez, J. (2004).Direct activation of the mitochondrial calcium uniporter by natural plant flavonoids.Biochem J,384(Pt1),19-24.doi:10.1042 / BJ20040990.

[0097]

[0124] It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

1. 1. A method for achieving at least one result selected from the group consisting of: (i) ameliorating a physiological condition associated with metabolic fatigue in one or more cells; (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells; and (iii) treating or preventing calcium deficiency / depletion disorders, comprising: A method comprising orally administering to an individual an effective amount of oleuropein or at least one of its metabolites.

2. 10. The method of claim 1, wherein at least a portion of the one or more cells is part of at least one body part selected from the group consisting of liver, kidney, brain, and skeletal muscle.

3. 3. The method of claim 1 or 2, wherein the physiological conditions associated with metabolic fatigue include muscle fatigue or weakness, lack of energy, lack of physical energy, lack of strength or weakness.

4. 4. The method of any one of claims 1 to 3, wherein the effective amount of oleuropein or at least one of its metabolites is orally administered daily for at least one week.

5. 5. The method of any one of claims 1 to 4, wherein the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfated forms thereof, derivatives thereof, and mixtures thereof.

6. 6. The method of any one of claims 1 to 5, wherein the effective amount of oleuropein or at least one of its metabolites is administered in a composition selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a nutraceutical, a beverage, a powdered nutritional product to be reconstituted with water or milk before ingestion, a food additive, a medicine, a drink, a pet food, and combinations thereof.

7. 7. The method of any one of claims 1 to 6, wherein the effective amount of oleuropein or at least one of its metabolites is administered in a composition further comprising calcium.

8. 8. The method of any one of claims 1 to 7, wherein the effective amount of oleuropein or at least one of its metabolites is administered in a food product further comprising an ingredient selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

9. 1. A method of treating or preventing a mitochondrial-associated disease or a condition associated with altered mitochondrial function in an individual in need of or at risk of such treatment, comprising: A method comprising orally administering to an individual in need of or at risk of said treatment an effective amount of oleuropein or at least one of its metabolites.

10. 10. The method of claim 9, wherein the mitochondrial-related disease or condition is selected from the group consisting of stress, physiological aging, obesity, metabolic slowing, metabolic syndrome, diabetes mellitus, complications from diabetes, hyperlipidemia, neurodegenerative disease, cognitive impairment, stress-induced or stress-related cognitive dysfunction, mood disorders, anxiety disorders, age-related neuronal cell death or dysfunction, chronic kidney disease, renal failure, trauma, infection, cancer, hearing loss, macular degeneration, myopathy, and dystrophies, and combinations thereof.

11. 11. The method of claim 9 or 10, wherein the effective amount of oleuropein or at least one of its metabolites is administered in a composition further comprising calcium.

12. A unit dosage form comprising oleuropein or at least one of its metabolites in an effective amount for at least one result selected from the group consisting of: (i) improving a physiological condition associated with metabolic fatigue in one or more cells, (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells, and (iii) treating or preventing calcium deficiency / depletion disorders.

13. 13. The unit dosage form of claim 12, wherein the physiological conditions associated with metabolic fatigue include muscle fatigue or weakness, lack of energy, lack of physical energy, lack of strength or weakness.

14. 14. The unit dosage form of claim 12 or 13, essentially comprising oleuropein or at least one of its metabolites.

15. 15. The unit dosage form of any one of claims 12 to 14, comprising an excipient and at least one of oleuropein or a metabolite thereof.

16. 16. The unit dosage form according to any one of claims 12 to 15, which essentially comprises calcium and said oleuropein or at least one of its metabolites.

17. 17. The unit dosage form of any one of claims 12 to 16, comprising an excipient, calcium, and at least one of oleuropein or a metabolite thereof.

18. 1. A method for producing a composition to achieve at least one result selected from the group consisting of: (i) ameliorating a physiological condition associated with metabolic fatigue in one or more cells; (ii) increasing mitochondrial energy and mitochondrial calcium uptake in one or more cells; and (iii) treating or preventing calcium deficiency / depletion disorders, comprising: A method comprising adding an effective amount of oleuropein or at least one of its metabolites to at least one ingredient selected from the group consisting of protein, carbohydrate, and fat.

19. 20. The method of claim 18, further comprising adding to the at least one ingredient a food additive selected from the group consisting of an acidulant, a thickener, a pH-adjusting buffer or agent, a chelating agent, a colorant, an emulsifier, an excipient, a flavoring, a mineral, an osmotic agent, a pharmaceutically acceptable carrier, a preservative, a stabilizer, a sugar, a sweetener, a modifier, a vitamin, a mineral, and combinations thereof.

20. 20. The method of claim 18 or 19, further comprising adding calcium to the at least one raw material.

Images