Compositions Comprising a Combination of Caffeine and Oleuropein or a Metabolite Thereof, and Their Use for Improving Muscle Function

Abstract

1. A combination of caffeine and at least one oleuropein or metabolite thereof, for use by oral administration to an individual in an effective amount to achieve at least one result which is: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) increasing mobility, and / or (vii) treating muscle disorders associated with calcium depletion or deficiency.

Description

[Technical field]

[0001] [Background technology]

[0001] The present disclosure relates generally to compositions and methods using a combination of caffeine and at least one oleuropein or metabolite thereof, and more specifically to compositions and methods that can improve, maintain, or reduce the decline of muscle function by combining caffeine and at least one oleuropein or metabolite thereof to enhance bioenergetics and mitochondrial function, enhance mitochondrial calcium transport, and thus enhance muscle contraction and performance.

[0002]

[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 the decline in muscle mass. Impaired muscle function is a highly predictive indicator for the occurrence of bedriddenness, disability, and death in old age. With the increase in the elderly population, the prevalence of sarcopenia is increasing, with 45% of the elderly population in the United States having moderate to severe symptoms. The direct and indirect medical costs attributable to sarcopenia in the United States are close to $19 billion. Therefore, the prevention and / or treatment of sarcopenia may have a significant impact on the health and quality of life of our society, and thus on the health-related economy. Unfortunately, the etiology and physiopathological mechanisms of sarcopenia remain poorly understood, making it difficult to develop effective measures for prevention or treatment.

[0003] [Summary of the Invention]

[0003] Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across the inner membrane, providing the signaling potential for the molecule. Furthermore, mitochondrial Ca2+ may play a role in regulating ATP production in mitochondria and contribute to orchestrating 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] The inventors have noted that aging involves a gradual decline in muscle function, capacity, and reactivity. For example, in a 50-year-old human, muscle area decreases by about 10%, and muscle strength decreases by about 15% per decade at ages 60 and 70, resulting in a decline of about 30%. Age-related loss of muscle mass is the cause of almost all of the decline in strength and vitality accompanied by increased fatigue in middle-aged and elderly people. This decline is due to the interrelated factors of lifestyle, structural changes in muscle, and metabolic changes.

[0005]

[0005] The present inventors have recognized this problem and addressed it with the surprising discovery that oleuropein and its metabolites are bioactives that activate mitochondrial calcium in combination with caffeine. Although calcium is essential for skeletal muscle contraction, there are very limited solutions to increase mitochondrial calcium uptake with natural bioactives to affect bioenergetics. Therefore, without being bound by theory, the present inventors believe that the combination of caffeine and at least one oleuropein or its metabolites can improve, maintain, or reduce the decline of muscle function by enhancing bioenergetics and mitochondrial function, enhancing mitochondrial calcium transport, and thus enhancing muscle contraction and muscle performance.

[0006]

[0006] 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 mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) increasing mobility, and (vii) treating or preventing (e.g., reducing the incidence and / or severity of) muscle disorders associated with calcium depletion or deficiency. The method comprises orally administering to an individual an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof.

[0007]

[0007] In one embodiment, the present invention relates to a method for reducing muscle fatigue in an individual participating in exercise, the exercise including at least one of 1) resistance exercise, 2) anaerobic exercise or repeated sprint type exercise, or 3) endurance exercise, the method including orally administering to the individual an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof.

[0008]

[0008] In one embodiment, the individual is selected from the group consisting of elderly subjects; aged subjects; subjects with muscle fatigue or muscle weakness; subjects with mobility impairment; frail subjects; pre-frail subjects; sarcopenic subjects; subjects recovering from pre-frailty, frailty, sarcopenia, or a mobility impairment; subjects undergoing physical rehabilitation (e.g., from injury to one or more of muscle, bone, ligament, or nervous system); sportsmen; and pets.

[0009]

[0009] In one embodiment, at least a portion of the muscle cells are part of a skeletal muscle selected from the group consisting of gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

[0010]

[0010] In one embodiment, a combination of caffeine and at least one oleuropein or metabolite thereof is administered orally daily for at least one week, preferably at least one month.

[0011]

[0011] In one embodiment, the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, glucuronidated forms thereof, sulfate forms thereof, derivatives thereof, and mixtures thereof.

[0012]

[0012] In one embodiment, the combination of caffeine and at least one oleuropein or metabolite thereof is administered in a composition selected from the group consisting of a food composition, a dietary supplement, a nutritional composition, a beverage, a nutraceutical, a powdered nutritional product to be reconstituted with water or milk prior to ingestion, a food additive, a medicine, a drink, a pet food, and combinations thereof.

[0013]

[0013] In one embodiment, caffeine and at least one oleuropein or metabolite thereof are administered together in the same composition.

[0014] In one embodiment, caffeine is administered separately in a different composition than at least one oleuropein or metabolite thereof.

[0015]

[0015] In one embodiment, caffeine and at least one oleuropein or metabolite thereof are administered together in a food product further comprising a component selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0016]

[0016] In another embodiment, the present disclosure provides a method for treating at least one condition selected from the group consisting of (i) at least one impairment of muscle function, muscle performance, or muscle strength, (ii) muscle fatigue or muscle weakness, (iii) pre-frailty, frailty, sarcopenia, or movement disorders, and (iv) muscle disorders associated with calcium depletion or deficiency in an individual in need of such treatment or for preventing (e.g., reducing the incidence and / or severity) in an individual at risk of such treatment. The method comprises orally administering to an individual in need or at risk an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof.

[0017]

[0017] In another embodiment, the present disclosure provides a unit dosage form comprising a combination of caffeine and at least one oleuropein or metabolite thereof, the unit dosage form comprising an amount of the combination effective for at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue, (vi) increasing mobility, and (vii) treating or preventing (e.g., reducing the incidence and / or severity of) muscle disorders associated with calcium depletion or deficiency.

[0018]

[0018] In one embodiment, the unit dosage form essentially comprises a combination of caffeine and at least one oleuropein or metabolite thereof.

[0019]

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

[0020]

[0020] 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 mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue or muscle weakness, (vi) improving mobility, and (vii) treating or preventing (e.g., reducing the incidence and / or severity of) muscle disorders associated with calcium depletion or deficiency. The method includes adding an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof to at least one raw material selected from the group consisting of protein, carbohydrate, and fat.

[0021]

[0021] In one embodiment, the method further includes adding a food additive selected from the group consisting of acidulants, thickeners, pH adjusting buffers or pH adjusting agents, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, penetrants, pharma- ceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, modifiers, vitamins, minerals, and combinations thereof to at least one raw material.

[0022]

[0022] Additional features and advantages are described herein, and will be apparent from the drawings and detailed description that follow. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows the effect of oleuropein aglycone (10 μM, grey), caffeine (5 mM, black), and the combination of 10 μM oleuropein aglycone + 5 mM caffeine on mitochondrial activation, measured as integrated mitochondrial calcium rise, in myotubes differentiated from C2C12 cells. Results are presented as mean ± SEM of n=29 experiments. * indicates statistical significance at P<0.05 (one-way ANOVA test). [Diagram 2] Figure 1 shows that oleuropein (10 μM) synergizes with caffeine (5 mM) to enhance mitochondrial calcium elevation in myotubes derived from C2C12 cells. The graph derived from the data shown in Figure 1 compares the theoretical additive effect of caffeine supplementation + oleuropein aglycone supplementation with the measured effect of simultaneous supplementation of the combination of caffeine + oleuropein aglycone on mitochondrial calcium elevation. The theoretical effect is compared to the measured effect to estimate synergy. Data are the average of 29 experiments. Results are expressed as mean ± SEM. * indicates statistical significance at P<0.05 (Student's t-test) for the difference between measured and theoretical values ​​in mitochondrial calcium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024]

[0025] definition

[0026] 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.

[0025]

[0027] All percentages stated herein are by total weight of the composition unless otherwise indicated. As used herein, "about," "approximately," and "substantially" are understood to refer to numbers within a certain range of numerical values, for example, within the range of -10% to +10% of the referenced number, preferably within the range of -5% to +5% of the referenced number, more preferably within the range of -1% to +1% of the referenced number, and most preferably within the range of -0.1% to +0.1% of the referenced number. All numerical ranges herein should be understood to include all integers or fractions within the range. Furthermore, these numerical ranges should be interpreted as supporting claims directed to any number or subset of numbers within the range. For example, a disclosure of 1 to 10 should be interpreted as corresponding to ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, etc.

[0026]

[0028] As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a metabolite" or "the metabolite" includes one metabolite, but also includes two or more metabolites.

[0027]

[0029] The terms "comprise", "comprises", and "comprising" should be interpreted as inclusive rather than exclusive. Similarly, the terms "include", "including", and "or" should all be interpreted as inclusive unless such interpretation is clearly prevented by the context. However, the compositions disclosed herein may not include elements not specifically disclosed herein. Thus, disclosure of an embodiment using the term "comprising" includes disclosure of an embodiment "consisting essentially of" the specified components, and an embodiment "consisting of" the specified components.

[0028]

[0030] As used herein, a "composition essentially comprising a combination of calcium and at least one oleuropein or metabolite thereof" does not include any additional compounds that affect mitochondrial calcium transport other than the combination of calcium and at least one oleuropein or metabolite thereof. In certain non-limiting embodiments, the composition consists of an excipient and a combination of calcium and at least one oleuropein or metabolite thereof.

[0029]

[0031] 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 oleuropein or a metabolite thereof" means "oleuropein" or "a metabolite of oleuropein" or "both oleuropein and a metabolite thereof".

[0030]

[0032] As used herein, the terms "example" and "such as," especially when followed by a list of terms, are merely exemplary and illustrative and should not be considered as exclusive or inclusive. 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.

[0031]

[0033] The terms "food," "food product," and "food composition" refer to a product or composition intended for ingestion by an individual, such as a human, and providing at least one nutrient to such an individual. The compositions of the present disclosure, including the many embodiments described herein, may comprise, consist of, or essentially comprise the elements disclosed herein, as well as any additional or optional ingredients, components, or elements described or not described herein that are useful in the diet.

[0032]

[0034] As used herein, the terms "treatment" and "treating" refer to the administration of a composition disclosed herein to a subject having a condition to attenuate, reduce or ameliorate at least one symptom associated with the condition and / or to slow, reduce or prevent the progression of the condition. The terms "treatment" and "treating" include both preventative or prophylactic treatments (treatments that prevent and / or slow the onset or progression of the targeted pathological condition or disorder) and curative, therapeutic or disease-modifying treatments, including, for example, therapeutic measures to cure, delay, reduce 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 as having a disease or medical condition. The terms "treatment" and "treating" do not necessarily mean treating the subject until they are cured. The terms "treatment" and "treating" also refer to maintaining and / or enhancing the health of an individual who is not afflicted with a disease but who may be susceptible to an unhealthy 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. As non-limiting examples, treatment can be performed by the patient, a caregiver, a doctor, a nurse, or another medical professional.

[0033]

[0035] Both human and animal treatments are within the scope of this disclosure. Preferably, the combination of caffeine and at least one oleuropein or metabolite thereof is administered in a serving or unit dosage form that provides a therapeutically or prophylactically effective amount of the combination.

[0034]

[0036] 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. Additionally, "prevention" includes reducing the risk, incidence, and / or severity of a condition or disorder.

[0035]

[0037] 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, reduces the symptoms, manages the progression of a disease, or provides a nutritional, physiological, or medical benefit to the individual.

[0036]

[0038] The relative terms "improved," "increased / enhanced / boosted," "enhanced / potentiated," and the like refer to the effect of a composition disclosed herein, i.e., a composition comprising an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof, compared to administration of an identical composition over the same period of time, except that it lacks caffeine or oleuropein / one of its metabolites.

[0037]

[0039] 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.

[0038]

[0040] "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, cows, and horses, and humans. When "animal", "mammal", or plurals thereof are used, these terms also apply to any animal in which, depending on the context, a demonstrated or intended effect can be demonstrated, for example, by the animal benefiting from improved mitochondrial calcium transport. Although the term "individual" or "subject" is often used herein to refer to a human, 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.

[0039]

[0041] 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.

[0040]

[0042] The term "old age" in the human context means 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" means a non-human subject that is likely to have reached 60% of its lifespan, in some embodiments at least 70%, at least 80%, or at least 90% of its lifespan. Assessment 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 affect lifespan positively or negatively. When assessing 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.

[0041]

[0043] The term "older adult" in the human context means a postnatal age of 45 years or older, preferably greater than 50 years, more preferably greater than 55 years, and includes elderly people.

[0042]

[0044] "Mobility" is the ability to move independently and safely from one place to another.

[0043]

[0045] Sarcopenia is defined as the age-related decline in muscle mass and function, including muscle strength and walking speed.

[0044]

[0046] As used herein, "frailty" is defined as a clinically discernible state of heightened vulnerability resulting from age-related declines in capacity and function across multiple biological systems, resulting in impaired ability to cope with routine or acute stressors. In the absence of established quantitative criteria, frailty has been operationally defined by Fried et al. as meeting three of five phenotypic criteria indicative of low energy: (1) weakness (grip strength in the bottom 20% of the population at baseline, adjusted for sex and body mass index), (2) low endurance and energy (self-reported fatigue associated with VO2 max), (3) slowness in walking speed (based on 15-foot walk time, bottom 20% of the population at baseline, adjusted for sex and height), (4) low physical activity (weighted score of kilocalorie intake per week at baseline, bottom of sex-specific physical activity quintile; e.g., <383 kcal / week for men and <270 kcal / week for women), and / or unintentional weight loss (10 pounds in the past year). Fried LP, Tangen CM, Walston J et al., "Frailty in older adults: evidence for a phenotype." J. Gerontol. A. Biol. Sci. Med. Sci. 56(3):M146-M156 (2001). The presence of one or two of these criteria identifies the pre-frailty stage as being at high risk for progression to frailty.

[0045]

[0047] "Muscle fatigue" refers to a decrease in the force of contraction in one or more muscles due to a lack of substrate within the muscle fibers and / or an accumulation of metabolic products within the muscle fibers that inhibit either the release of calcium or the ability of calcium to stimulate muscle contraction.

[0046]

[0048] "Weakness" is a condition in which the force exerted by a muscle is less than what it could be. The US Medical Research Council's grading system for muscle strength is widely used to identify muscle weakness and its severity. In particular, an examiner evaluates the patient's ability to move a muscle against resistance provided by an examiner who has a sense of what is expected to be in the normal range. Evaluations may vary between patients, depending on the subject's underlying size and condition, etc. A well-trained athlete can expect different results than a smaller, less active, or less physically fit individual. Expected muscle strength must also be adjusted for the degree of atrophy in patients with wasting diseases.

[0047]

[0049] The patient's attempts are rated on a scale of 0 to 5. As used herein, "muscle weakness" refers to any of grades 0 to 4. Grade 5: Normal muscle contraction against maximum resistance. Grade 4: Muscle strength is reduced, but the joint can be moved by muscle contraction against resistance. Grade 3: Strength is further reduced and the patient is able to move the joint against gravity alone with no resistance provided by the examiner (e.g., starting with the arm at the side, the elbow can be moved from full extension to full flexion). Grade 2: The muscle can only be moved in the absence of resistance from gravity, e.g. the elbow can only be fully flexed if the arm is held in a horizontal plane. Grade 1: A trace or flicker movement is seen or felt in the muscle, or fasciculations are observed in the muscle. Grade 0: No movement is observed.

[0048]

[0050] As used herein, an "athlete" is an individual who participates in at least one of the following: 1) resistance exercise, 2) anaerobic or repeated sprint type exercise, or 3) endurance exercise.

[0049]

[0051] Resistance exercise is when the subject performs instantaneous transfer of weight and long rest periods, and is driven primarily by the creatine phosphate and glycolysis energy systems. Resistance exercise can produce energy quickly, but subjects fatigue quickly. The main adaptation is to increase muscle mass (hypertrophy) by increasing muscle cross-sectional area through repeated weight lifting training. Hakkinen K.1989.Neuromuscular and hormonal adaptations during strength and powher training.J.Sports Med.Phys.Fitness.29:9-26;and Hakkinen K.et.al.1987.Relationships between training vulolume,physical performance capacity,and serum hormone concentrations during prolonged training in elite wheight lifters.Int.J.Sports Med.8 Suppl 1:61-65.

[0050]

[0052] Repeated sprint-type training is anaerobic, involves high-intensity exercise with limited recovery periods, and involves almost pure carbohydrate metabolism with extensive breakdown in muscle glycogen (energy production by glycolysis). During anaerobic energy production situations, such as high-intensity speed training or sports involving repeated sprints, increased muscle load is achieved by increased firing of type IIa muscle fibers. Finally, at very high workloads, type IIb glycolytic muscle fibers are activated and high demand energy supply is maintained by anaerobic energy supply. However, during these situations, the high rate of anaerobic energy production exceeds the rate at which it can be aerobically oxidized in mitochondria, thus leading to the extreme levels of lactate production seen in these types of training situations. Spriet LL, Howlett RA, and Heigenhauser G J. 2000. An enzymatic approach to lactate production in human skeletal muscle during exercise. Med. Sci. Sports Exerc. 32: 756-763.

[0051]

[0053] Endurance training is characterized by an individual engaging in low-intensity training for extended periods of time (e.g., more than 15 minutes). The energy systems exhibited in endurance training include the aerobic system, which produces the required energy when sufficient oxygen is present, primarily using aerobic metabolism of fats and carbohydrates in mitochondria. Major adaptations include increased muscle glycogen stores and increased glycogen sparing at sub-maximal workloads due to increased fat oxidation, enhanced lactate kinetics, and morphological changes including increased type I muscle fibers per muscle area and increased capillary and mitochondrial density. Holloszy JO, and Coyle E F. 1984. Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J. Appl. Physiol. 56:831-838; exercise.Ann.NYAcad.Sci.301:440-450.

[0052]

[0054] The terms "serving" or "unit dosage form" as used herein are interchangeable and refer to physically separate units suitable as a unitary dose for human and animal subjects, each unit containing a predetermined amount of a composition comprising a combination of calcium and at least one oleuropein or its metabolites as disclosed herein, preferably with a pharma-ceutically acceptable diluent, carrier, or vehicle, in an amount sufficient to produce a desired effect. The specifications of the unit dosage form depend on the specific compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In one embodiment, the unit dosage form can be a predetermined amount of liquid contained in a container such as a bottle.

[0053]

[0055] An "oral nutritional supplement" or "ONS" is a composition that contains at least one macronutrient and / or at least one micronutrient, e.g., in a sterile liquid, semisolid, or powder form, and is 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, the ONS can be in a liquid form that can be consumed without the addition of additional liquid, e.g., the amount of liquid is one serving of the composition.

[0054]

[0056] As used herein, "incomplete nutrition" refers to a nutritional product that preferably does not contain sufficient levels of macronutrients (protein, fat and carbohydrate) 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 its nutritional requirements from a complete nutritional composition.

[0055]

[0057] "Kit" means that the components of the kit are physically associated in or with one or more containers and are considered a single unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, cartons, bottles, packaging of any type or design or material, overwraps, shrink wraps, affixed components (e.g., stapled or glued), or combinations thereof.

[0056]

[0058] Embodiment

[0059] Another aspect of the present disclosure is a method for achieving at least one result selected from the group consisting of: (i) improving mitochondrial calcium uptake in muscle cells, (ii) improving calcium utilization in muscle cells, (iii) increasing mitochondrial energy in muscle cells, (iv) improving at least one of muscle function, muscle performance, or muscle strength, (v) reducing muscle fatigue or muscle weakness, (vi) improving mobility, and (vii) treating or preventing (e.g., reducing the incidence and / or severity of) muscle disorders associated with calcium depletion or deficiency. The method comprises orally administering to an individual an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof.

[0057]

[0060] Another aspect of the present disclosure is a method for treating or preventing in an individual at risk of at least one condition selected from the group consisting of: (i) at least one impairment of muscle function, muscle performance, or muscle strength; (ii) muscle fatigue or muscle weakness; (iii) pre-frailty, frailty, sarcopenia, or movement disorders; and (iv) muscle disorders associated with calcium depletion or deficiency, comprising orally administering to an individual at risk or in need of treatment an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof.

[0058]

[0061] This may also result in improved vitality and / or energy in the individual.

[0059] In another embodiment, the reduction in muscle fatigue is in an individual participating in exercise, the exercise being at least one of 1) resistance exercise, 2) anaerobic exercise or repeated sprint type exercise, or 3) endurance exercise, and the method comprises orally administering to the individual an effective amount of a combination of caffeine and at least one oleuropein or its metabolite. The combination of caffeine and at least one oleuropein or its metabolite is administered to the individual before exercise, and / or during exercise, and / or after exercise, preferably less than 2 hours before exercise, and / or during exercise, and / or less than 2 hours after exercise. In a more preferred embodiment, the combination of caffeine and at least one oleuropein or its metabolite is administered to the individual less than 1 hour before exercise.

[0060]

[0062] The effective amount of the combination of caffeine and at least one oleuropein or metabolite thereof varies depending on the specific composition, the age and condition of the recipient, and the specific disorder or disease being treated. However, in a general embodiment, an individual may be administered 0.001 mg to 1.0 g of at least one oleuropein or metabolite thereof per day, preferably 0.01 mg to 0.9 g of at least one oleuropein or metabolite thereof per day, more preferably 0.1 mg to 750 mg of at least one oleuropein or metabolite thereof per day, more preferably 0.5 mg to 500 mg of at least one oleuropein or metabolite thereof per day, and most preferably 1.0 mg to 200 mg of at least one oleuropein or metabolite thereof per day.

[0061]

[0063] 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 kernels of a plant belonging to the family Oleaceae, preferably from a plant belonging to the family Oleaceae, such as olive (olive tree), plants of the genus Ligustrum, plants of the genus Syringa, plants of the genus Fraximus, plants of the genus Jasminum, plants of the genus Osmanthus, etc. Additionally or alternatively, at least a portion of the oleuropein can be obtained by chemical synthesis.

[0062]

[0064] Non-limiting examples of suitable metabolic products of oleuropein include oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovanillyl alcohol, and mixtures thereof.

[0063]

[0065] In some embodiments, at least one oleuropein or metabolite thereof is the only polyphenol in the composition and / or the only polyphenol administered to the individual.

[0064]

[0066] The effective amount of caffeine will also vary depending on the specific composition, the age and condition of the recipient, and the specific disorder or disease being treated. However, in general embodiments, up to 400 mg of caffeine may be administered to an individual per day, preferably from about 20 mg to about 380 mg, and more preferably from about 40 to about 300 mg per day. Alternatively, caffeine may also be present in the composition in an amount of about 0.05 to 0.3 g / serving, about 0.1 to 0.2 g / serving, about 0.125 to 0.175 g / serving, or about 0.14 g / serving.

[0065]

[0067] In some embodiments, the combination of caffeine and at least one of oleuropein or its metabolites is administered to an individual selected from the group consisting of: aged subjects; elderly subjects; subjects with muscle fatigue or muscle weakness; subjects with movement disorders; subjects that are frail; subjects that are pre-frail; subjects with sarcopenia; subjects recovering from pre-frailty, frailty, sarcopenia, or movement disorders; subjects undergoing physical rehabilitation (e.g., from injury to one or more of muscle, bone, ligament, or nervous system); sportsmen; and pets.In some embodiments, the individual is healthy.In some embodiments, the individual has sarcopenia, frailty, muscle fatigue or muscle weakness, or one or more impairments of muscle function, muscle performance, or muscle strength, but is optionally otherwise healthy.

[0066]

[0068] For example, a combination of caffeine and at least one oleuropein or metabolite thereof can be administered to an athlete before, during, and / or after exercise, for example, less than 2 hours before exercise, or less than 1 hour before exercise and less than 2 hours after exercise, or less than 1 hour after exercise.

[0067]

[0069] In one embodiment, at least a portion of the muscle cells are part of a skeletal muscle selected from the group consisting of gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

[0068]

[0070] The combination of caffeine and at least one oleuropein or its metabolites can be administered in any composition suitable for human and / or animal consumption.In a preferred embodiment, the combination of caffeine and at least one oleuropein or its metabolites is administered to an individual orally or enterally (e.g., tube feeding).For example, the combination of caffeine and at least one oleuropein or its metabolites can be administered to an individual in a beverage, food product, capsule, tablet, powder, or suspension.

[0069]

[0071] 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 prior to ingestion, food additives, medicaments, drinks, pet foods, and combinations thereof.

[0070]

[0072] Food products according to the invention include, by way of example, dairy products, e.g. fermented dairy products such as yoghurt, buttermilk and the like; ice cream; condensed milk; milk; dairy creamers; flavoured milk drinks; whey-based drinks; toppings; coffee creamers; chocolate; cheese-based products; soups; sauces; purees; dressings; puddings; custards; baby foods; nutritional formulas, e.g. complete nutritional formulas for infants, children, teenagers, adults, the elderly or the seriously ill; cereals and cereal bars.

[0071]

[0073] The 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, fruit and / or vegetable juices.

[0072]

[0074] The combination of caffeine and at least one oleuropein or metabolite thereof may be administered in a food product further comprising a component selected from the group consisting of protein, carbohydrate, fat, and mixtures thereof.

[0073]

[0075] In cases where oral or enteral administration is not possible or advisable, the compositions may be administered parenterally.

[0074]

[0076] Preferably, the muscle function that can be improved by the methods disclosed herein includes a property selected from the group consisting of muscle strength, walking speed, and combinations thereof. Muscle function is typically defined as strength per unit of appendicular skeletal muscle mass or per muscle volume.

[0075]

[0077] Non-limiting examples of muscle disorders associated with calcium depletion or deficiency that can be treated by the methods disclosed herein include muscular dystrophies, congenital core myopathies, and mitochondrial myopathies. Specific non-limiting examples include Barth syndrome; chronic progressive external ophthalmoplegia (cPEO); Kearns-Sayre syndrome (KSS); Leigh syndrome; mitochondrial DNA depletion syndrome (MDDS); mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS); mitochondrial neurogastrointestinal encephalomyopathy (MINGIE); myoclonic epilepsy with ragged-red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); and Pearson syndrome.

[0076]

[0078] The individual may be at risk for a disorder or condition (e.g., sarcopenia, frailty, muscle fatigue or weakness, or one or more disorders of muscle function, muscle performance, or muscle strength), in which case the effective amount of the composition is a prophylactically effective dose, or the individual may have a disorder or condition, in which case the effective amount of the composition is a therapeutically effective dose. In some embodiments, the method includes identifying the individual as having or at risk for the condition prior to administration.

[0077]

[0079] In another embodiment, the present disclosure provides a method for treating or preventing movement disorders in elderly people. The method comprises orally administering to elderly people an effective amount of a combination of caffeine and at least one oleuropein or its metabolite. The elderly person may be an elderly individual. In some embodiments, the elderly person with sarcopenia, osteoporosis, osteoarthritis, recovering from malnutrition, at risk of malnutrition, undergoing rehabilitation, scheduled to undergo rehabilitation within the next year, and combinations thereof, has a condition selected from the group consisting of frailty, pre-frailty, and sarcopenia.

[0078]

[0080] The composition can be administered to a middle-aged or elderly person in an amount sufficient to at least partially reduce the risk of developing frailty or sarcopenia if the condition has not yet manifested in the individual, and / or at least partially reduce the severity of pre-frailty, frailty, sarcopenia, or mobility impairment. Such an amount is defined as a "prophylactically effective dose." Again, the exact amount depends on a number of factors related to the individual, such as weight, health status, and the extent to which muscle function (e.g., muscle strength, walking speed, etc.) has been reduced.

[0079]

[0081] In one embodiment, the combination of caffeine and at least one oleuropein or metabolite thereof is administered to an individual for a period of at least 1 month, preferably at least 2 months, more preferably at least 3, 4, 5 or 6 months, most preferably at least 1 year.During the period, the combination of caffeine and at least one oleuropein or metabolite thereof can be administered to an individual at least 1 day per week, preferably at least 2 days per week, more preferably at least 3, 4, 5 or 6 days per week, most preferably 7 days per week.The combination of caffeine and at least one oleuropein or metabolite thereof can be administered in a single dose per day or in multiple separate doses per day.

[0080]

[0082] The above dosing examples do not require uninterrupted daily administration. Instead, there may be several short interruptions in administration, such as a 2-4 day break during the administration period. The ideal duration of administration of the compositions of the present invention can be determined by one of skill in the art.

[0081]

[0083] In one embodiment, caffeine and at least one oleuropein or metabolite thereof can be administered in the same composition, for example, in a unit dosage form containing both caffeine and at least one oleuropein or metabolite thereof.

[0082]

[0084] In an alternative embodiment, caffeine and at least one oleuropein or metabolite thereof can be administered consecutively in separate compositions. The term "consecutively" means that caffeine and at least one oleuropein or metabolite thereof are administered sequentially, such that at a first time, at least one oleuropein or metabolite thereof is administered without caffeine, and at a second time (before or after the first time), caffeine is administered without at least one oleuropein or metabolite thereof. The time between consecutive administrations can be, for example, a second or a few seconds, minutes, or hours on the same day; a day or a few days, or a few weeks on the same month; or a month or a few months on the same year.

[0083]

[0085] 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 mitochondrial calcium uptake in muscle cells; (ii) improving calcium utilization in muscle cells; (iii) increasing mitochondrial energy in muscle cells; (iv) improving at least one of muscle function, muscle performance, or muscle strength; (v) reducing muscle fatigue; (vi) increasing mobility; and (vii) treating muscle disorders associated with calcium depletion or deficiency.

[0084]

[0086] The method includes adding a combination of caffeine and at least one oleuropein or metabolite thereof to a raw material selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof. The composition (e.g., food product) can be made before 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 before administration, preferably less than 15 minutes before administration, more preferably less than 10 minutes before administration, and most preferably less than 5 minutes before administration).

[0085]

[0087] The composition can include an effective amount of a combination of caffeine and at least one oleuropein or metabolite thereof, for example, a single serving or dose of the composition can include an effective amount of the combination, and a package can include one or more servings or doses.

[0086]

[0088] The composition may include a food additive selected from the group consisting of an acidulant, a thickener, a pH adjusting buffer or pH adjusting agent, a chelating agent, a coloring agent, an emulsifier, an excipient, a flavoring agent, a mineral, an osmotic agent, a pharma- ceutically acceptable carrier, a preservative, a stabilizer, a sugar, a sweetener, a modifier, a vitamin, a mineral, and combinations thereof.

[0087]

[0089] In addition to the combination of caffeine and at least one oleuropein or its metabolites, the composition can further comprise a protein source of animal or plant origin, 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; protein from legumes, cereals and grains; and combinations thereof.In addition or alternatively, the protein source can comprise protein from nuts and / or seeds.

[0088]

[0090] The protein source preferably comprises whey protein. The whey protein may be unhydrolyzed or hydrolyzed whey protein. 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 with caseino-glycomacropeptide removed), whey protein fractions, and any combination thereof. In a preferred embodiment, the whey protein comprises whey protein isolate and / or denatured sweet whey.

[0089]

[0091] As mentioned above, the protein source may be of animal or vegetable 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 milk, and preferably as casein micelles.

[0090]

[0092] 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 free form and / or leucine bound as a peptide and / or protein, such as milk 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 leucine per kg body weight, preferably 0.02-0.035 g leucine per kg body weight. Such doses are particularly suitable for complete nutritional compositions, but the skilled artisan will readily recognize how to adjust the dose for oral nutritional supplements (ONS).

[0091]

[0093] One or more other minerals may be used in the composition. Non-limiting examples of suitable minerals include calcium, boron, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, selenium, silicon, tin, vanadium, zinc, or combinations thereof.

[0092]

[0094] One or more other vitamins can be used in the composition, which are added to any one of them.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, or pyridoxamine, or pyridoxine hydrochloride), vitamin B7 (biotin), vitamin B9 (folic acid), and vitamin B12 (various cobalamins; typically cyanocobalamin in vitamin supplements), vitamin C, vitamin D, vitamin E, vitamin K, folic acid, and biotin), and combinations thereof."Vitamin" includes compounds such as provitamins, their derivatives, and their analogs, which are naturally obtained from plant and animal foods or are synthesized.

[0093]

[0095] 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 acts as a natural bulking and laxative agent. Dietary fiber may be soluble or insoluble, with blends 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.

[0094]

[0096] One or more food grade emulsifiers may be incorporated into the compositions, such as diacetyl tartaric acid esters of mono- and diglycerides, lecithin, and / or mono- and diglycerides. Suitable salts and stabilizers may also be included. EXAMPLES

[0095]

[0098] The following non-limiting examples provide experimental data supporting the compositions and methods disclosed herein.

[0096]

[0099] Example 1 To test the synergistic effect of the combination of oleuropein + caffeine on mitochondrial calcium elevation, we measured mitochondrial calcium elevation in myotubes differentiated from C2C12 cells. C2C12 cells were purchased from ATCC. C2C12 cells were seeded in 96-well plates at a density of 8000 cells / well in DMEM high glucose (Gibco) + 10% fetal bovine serum. Myotubes were differentiated from C2C12 cells by growing the cells for 4 days in DMEM containing 2% horse serum. Mitochondrial calcium measurements were performed using myotubes infected with an adenovirus (Sirion biotech) expressing mitochondrial modified aequorin, a calcium sensor targeted in mitochondria (Montero et al., 2004). For aequorin reconstitution, 24 hours after infection, myotubes were cultured in standard medium (145 mM NaCl, 5 mM KCl, 1 mM MgCl) supplemented with 1 μM wild-type coelenterazine. 2 , 1 mM CaCl 2 , 10 mM glucose, and 10 mM Hepes, pH 7.4) for 2 h at room temperature (22 ± C). For treatment, compounds were added directly to cell or myotube cultures 2 h prior to measurement. Luminescence was measured on a FLIPR Tetra Aequorin (Molecular Devices). Conversion of luminescence data to calcium concentration (Calibration) was performed using an algorithm as previously described (Alvarez & Montero, 2002). Quantification was performed using a custom module analysis based on Excel (Microsoft) and GhaphPad Prism 7.02 (GhaphPad) software.

[0097] As shown in FIG. 1, co-supplementation with oleuropein aglycone and caffeine results in greater mitochondrial activation (measured as mitochondrial calcium elevation) than the effect of each compound alone.

[0098] To calculate the synergistic effect of oleuropein aglycone + caffeine, the theoretical effect of the sum of the two individual compounds (oleuropein aglycone and caffeine) was compared to the measured effect of the combination, as shown in main Figure 2. As shown in Figure 2, oleuropein synergizes with caffeine to promote mitochondrial calcium elevation during stimulation.

[0099]

[0100] References

[0101] Glancy, B. and RSBalaban (2012). “Role of mitochondrial Ca2+ in the regulation of cellular energetics.” Biochemistry 51(14):2959-2973).

[0102] Alvarez, J., & Montero, M. (2002). Measuring[Ca2+] in the endoplasmic reticulum whith aequorin. Cell Calcium, 32(5-6), 251-260.

[0103] 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.

[0100]

[0104] 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. Accordingly, such changes and modifications are intended to be encompassed by the appended claims.

Claims

1. A composition for use in a method for achieving at least one outcome selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved calcium utilization in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improved at least one of muscle function, muscle performance, or muscle strength, (v) reduced muscle fatigue, (vi) improved mobility, and (vii) treatment of muscle disorders associated with calcium depletion or deficiency, The method includes orally administering the composition to an individual, The composition comprises an effective amount of a combination of caffeine and at least one oleuropein or its metabolite.

2. The composition according to claim 1, wherein the individual is selected from the group consisting of an elderly subject, an older subject, a subject with muscle fatigue or muscle weakness, a subject with motor impairment, a frail subject, a pre-frail subject, a subject with sarcopenia, a subject recovering from pre-frailty, frailty, sarcopenia, or motor impairment, a subject undergoing physical rehabilitation, an athlete, and a pet.

3. The composition according to claim 1, wherein the reduction in muscle fatigue is observed in individuals participating in exercise, and the exercise comprises at least one of the following: 1) resistance exercise, 2) anaerobic exercise or repetitive sprinting exercise, or 3) endurance exercise.

4. The composition according to claim 3, wherein the composition is administered to the individual before and / or during and / or after the exercise.

5. The composition according to claim 1, wherein at least a portion of the muscle cells is a part of a skeletal muscle selected from the group consisting of the gastrocnemius, tibialis, soleus, extensor digitorum longus (EDL), biceps femoris, semitendinosus, semimembranosus, gluteus maximus, and combinations thereof.

6. The composition according to claim 1, administered daily for at least one week.

7. The composition according to claim 1, wherein the metabolite of oleuropein is selected from the group consisting of oleuropein aglycone, hydroxytyrosol, homovanillyl alcohol, isohomovannillyl alcohol, glucuronide conjugates thereof, sulfate forms thereof, derivatives thereof, and mixtures thereof.

8. The composition according to claim 1, wherein the composition is administered in a composition selected from the group consisting of food compositions, dietary supplements, nutritional compositions, beverages, nutraceuticals, powdered nutritional products that are reconstituted with water or milk before ingestion, food additives, pharmaceuticals, drinks, pet food, and combinations thereof.

9. The composition according to claim 1, wherein the caffeine and at least one oleuropein or its metabolite are administered together in the same composition.

10. The composition according to claim 1, wherein the caffeine is administered separately in a composition different from the at least one oleuropein or its metabolite.

11. The composition according to claim 1, wherein the composition is administered together with a food product further comprising a component selected from the group consisting of proteins, carbohydrates, fats, and mixtures thereof.

12. A composition for use in a method of treating or preventing in an individual at risk of treating at least one condition selected from the group consisting of (i) impairment of at least one of muscle function, muscle performance, or muscle strength, (ii) muscle fatigue or weakness, (iii) pre-frailty, frailty, sarcopenia, or motor impairment, and (iv) muscle disorders associated with calcium depletion or deficiency, The method includes orally administering the composition to the individual who requires the treatment or is at risk of the treatment, The composition comprises an effective amount of a combination of caffeine and at least one oleuropein or its metabolite.

13. A unit dosage form comprising a combination of caffeine and at least one oleuropein or its metabolite, wherein the unit dosage form comprises an amount of the combination effective for at least one outcome selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved calcium utilization in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improvement of at least one of muscle function, muscle performance, or muscle strength, (v) reduced muscle fatigue, (vi) improved mobility, and (vii) treatment of muscle disorders associated with calcium depletion or deficiency.

14. The unit dosage form according to claim 13, which essentially comprises the combination of caffeine and at least one oleuropein or its metabolite.

15. The unit dosage form according to claim 13 or 14, comprising an excipient and the combination of caffeine and at least one oleuropein or its metabolite.

16. A method for producing a composition to achieve at least one result selected from the group consisting of (i) improved mitochondrial calcium uptake in muscle cells, (ii) improved calcium utilization in muscle cells, (iii) increased mitochondrial energy in muscle cells, (iv) improved at least one of muscle function, muscle performance, or muscle strength, (v) reduced muscle fatigue or weakness, (vi) improved mobility, and (vii) treatment of muscle disorders associated with calcium depletion or deficiency, comprising adding an effective amount of a combination of caffeine and at least one oleuropein or its metabolite to at least one raw material selected from the group consisting of proteins, carbohydrates, and fats.

17. The method according to claim 16, further comprising adding to at least one of the raw materials a food additive selected from the group consisting of acidulants, thickeners, pH-adjusting buffers or pH adjusters, chelating agents, colorants, emulsifiers, excipients, flavorings, minerals, penetrating agents, pharmaceutically acceptable carriers, preservatives, stabilizers, sugars, sweeteners, seasonings, vitamins, minerals, and combinations thereof.

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