FIELD OF THE INVENTION

[0001] The present invention relates to methods for promoting muscle regeneration in a subject, and to such methods employing nutritional compositions.

BACKGROUND OF THE INVENTION

[0002] Skeletal muscle (also referred to herein simply as “muscle”) is a highly dynamic and adaptive tissue and is implicated in multiple mechanical, physiological and metabolomic processes including movement, metabolism and homeostasis. Skeletal muscle is composed of large, multinucleated cells called myofibers. Adult skeletal muscle fiber number is set in-utero and adult fibers are terminally differentiated, i.e. , are incapable of division. Despite these phenomena, adult skeletal muscle is highly adaptable, responding to the soluble and biophysical cues that it encounters on a daily basis, referred to as skeletal muscle plasticity. [0003] It is important to increase muscle mass, not only for athletes but also for many other people. For example, it may be desirable to increase muscle mass in the elderly, as aging can reduce muscle mass, in individuals who suffer chronic diseases which result in muscle wasting, for example, myopathies, obesity, diabetes, cancer, cachexia, and viral infections, or those undergoing therapies for a disease such as chemotherapy, and in immobilized individuals, for example, those experiencing hospitalization, bed rest, or broken limbs.

[0004] Anaerobic exercise, such as resistance training, is one of the most effective strategies to promote muscle hypertrophy, i.e., an increase in muscle mass and volume involving an increase in muscle fiber cross-sectional area. It is widely accepted that the main mechanism for inducing muscle hypertrophy by exercise training is to increase muscle protein synthesis in muscle fibers. Evidence also supports the involvement of muscle satellite cells in the process of training-induced muscle hypertrophy. Muscle satellite cells, located between the basal lamina and sarcolemma of the muscle, are precursor cells with the potential to differentiate into mature muscle cells. These myogenic progenitor cells are normally in a quiescent state, although once stimulated, they become “activated” for the generation of new muscle fiber cells. Activated satellite cells proliferate and then differentiate into newly formed or nascent myotubes to become fully mature and functional myofibers, ultimately regenerating muscle tissue.

[0005] Muscle damage is known to be a potent stimulator of satellite cells, inducing them to convert from the quiescent sate to the activated state. Muscle damage in the form of microtrauma is present in numerous physiological conditions such as exercise-induced muscle damage (EIMD), which is frequently observed following physical activity and/or strenuous exercise and especially when eccentric contractions are incorporated in exercise. Moreover, numerous catabolic and muscle wasting chronic conditions as noted previously result in a chronic state of muscle microtrauma. Hence, muscle regeneration is one of the most important homeostatic processes of adult tissue and, as such, must be finely regulated to guarantee functional recovery and to avoid muscle alteration and diseases. The impairment of skeletal muscle regenerative potential characterizes a suite of physiopathologic conditions severely affecting human health.

[0006] Currently, the clinical treatment of muscle damage relies upon conventional therapies of rest, ice, and anti-inflammatory medications, especially in athletes. In addition, supplementation with vitamins (predominantly vitamins C and E) has been extensively utilized with the objective of altering redox status and improving performance in athletes. It would be advantageous however to develop a nutritional approach for muscle regeneration that provides specific nutritional ingredients that can promote muscle differentiation and proliferation. A nutritional approach is desirable as it can be easily administered, thereby improving outcomes. Beta-hydroxy beta-methylbutyrate (HMB) has been described as a promising agent for enhancing increases in the strength of muscle, aerobic performance, and resistance to fatigue. A major advantage of the use of HMB as a nutritional supplement is associated with its anti- catabolic action in muscle. Furthermore, several studies have showed that flavonoids, present in different plants, for example, quercetin (present in red wine, onions, green tea, kale, apples, and berries) and resveratrol (present in grapes, blueberries, raspberries, mulberries, and peanuts), act on protein catabolism and muscle function, mainly through their antioxidant and antiinflammatory properties. Notwithstanding these known effects of HMB and the indicated flavonoids, a need exists for conveniently providing improvements in promoting muscle regeneration.

SUMMARY OF THE INVENTION

[0007] Accordingly, it is an object of the invention to provide a convenient means for promoting muscle regeneration in an individual in need thereof.

[0008] In one embodiment, the invention is directed to methods for promoting muscle regeneration in a subject. The methods comprise administering beta-hydroxy betamethylbutyrate (HMB) and at least one citrus flavonoid to the subject.

[0009] In a more specific embodiment, the invention is directed to methods for promoting muscle regeneration by administering a nutritional composition comprising protein, fat, carbohydrate, HMB, and at least one citrus flavonoid.

[00010] The methods of the invention are advantageous in promoting muscle regeneration and maintaining or increasing muscle mass and/or volume in the subject.

[00011] Additional aspects and advantages of the present invention will be more fully described in view of the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[00012] The drawings illustrate certain aspects of embodiments of the invention, in which: [00013] Fig. 1 shows the effects of HMB and flavonoid-containing citrus extract (orange peel extract, OPE), individually and in combination, on creatine kinase expression, which is involved in muscle cell differentiation, in L6.C11 rat skeletal muscle myoblasts, as described in the Example; [00014] Fig. 2 shows the effects of HMB and hesperidin, individually and in combination, on creatine kinase expression in L6.C11 rat skeletal muscle myoblasts, as described in the Example;

[00015] Fig. 3 shows the effects of HMB and hesperetin, individually and in combination, on creatine kinase expression in L6.C11 rat skeletal muscle myoblasts, as described in the Example;

[00016] Fig. 4 shows the effects of HMB and flavonoid-containing citrus extract, OPE, individually and in combination, on protein synthesis in L6.C11 rat skeletal muscle myoblasts, as described in the Example;

[00017] Fig. 5 shows the effects of HMB and hesperidin, individually and in combination, on protein synthesis in L6.C11 rat skeletal muscle myoblasts, as described in the Example; and [00018] Fig. 6 shows the effects of HMB and hesperetin, individually and in combination, on protein synthesis in L6.C11 rat skeletal muscle myoblasts, as described in the Example.

DETAILED DESCRIPTION

[00019] While the general inventive concepts are susceptible of embodiment in many different forms, described herein in detail are specific embodiments of the invention, with the understanding that the present disclosure is to be considered as an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated and described herein.

[00020] All percentages, parts and ratios as used herein, are by weight of the total composition, unless otherwise specified. All such weights as they pertain to listed ingredients are based on the active level and, therefore, do not include solvents or byproducts that may be included in commercially available materials, unless otherwise specified.

[00021] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting the disclosure as a whole. Unless otherwise specified, “a, an,” “the,” and “at least one” are used interchangeably. Furthermore, as used in the description and the appended claims, the singular forms “a,” “an,” and “the” are inclusive of their plural forms, unless the context clearly indicates otherwise.

[00022] Throughout this specification, when a range of values is defined with respect to a particular characteristic of the present invention, the present invention relates to and explicitly incorporates every specific subrange therein. Additionally, throughout this specification, when a group of substances is defined with respect to a particular characteristic of the present invention, the present invention relates to and explicitly incorporates every specific subgroup therein. Any specified range or group is to be understood as a shorthand way of referring to every member of a range or group individually as well as every possible subrange or subgroup encompassed therein.

[00023] The methods and nutritional compositions described herein may comprise, consist of, or consist essentially of the essential steps and elements, respectively, as described herein, as well as any additional or optional steps and elements, respectively, described herein. Any combination of method or process steps as used herein may be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.

[00024] The various embodiments of the nutritional compositions employed in the methods of the invention may also be substantially free of any optional or selected ingredient or feature described herein, provided that the remaining nutritional composition still contains all of the required ingredients or features as described herein. In this context, and unless otherwise specified, the term “substantially free” means that the selected nutritional composition contains less than a functional amount of the optional ingredient, typically less than 1%, including less than 0.5%, including less than 0.1%, and also including zero percent, by weight, of such optional or selected essential ingredient. [00025] Unless otherwise indicated herein, all exemplary embodiments, sub-embodiments, specific embodiments and optional embodiments are respective exemplary embodiments, subembodiments, specific embodiments and optional embodiments to all embodiments described herein.

[00026] In a first embodiment, the invention is directed to methods for promoting muscle regeneration in a subject. Without being limited by theory, it is believed that the present methods promote muscle regeneration by increasing myogenic differentiation (see the Example and Figs. 1-3) and myotube maturation/hypertrophy (see the Example and Figs. 4-6), thereby increasing and growing muscle cells. The method of the present invention is based on upon discovery that the combination of HMB and citrus flavonoids shows a synergetic effect on enhancing both muscle cell differentiation and maturation in a preclinical model. Both mechanisms promote muscle regeneration capacity in those situations where it can be compromised. The muscle regeneration promoted by the present methods will therefore typically result in maintaining muscle and/or muscle hypertrophy, i.e. , an increase in muscle fiber cross-sectional area or, more specifically, increased muscle mass and/or volume. The methods conveniently provide such improvements by administering HMB and at least one citrus flavonoid to the subject. In a specific embodiment, the invention is directed methods of promoting muscle regeneration by administering nutritional compositions comprising HMB and at least one citrus flavonoid, providing a convenient means for conducting the inventive methods.

[00027] In specific embodiments, the subject is in need of muscle regeneration promotion. For example, the subject may have experienced muscle damage. Thus, in one embodiment, the subject has experienced exercise-induced muscle damage, for example, following physical activity and/or strenuous exercise. Alternatively, or in addition, the subject may be experiencing muscle damage as a result of aging, as aging is known to reduce muscle mass. In specific embodiments, the subject is of at least 40 years of age, of at least 50 years of age, of at least 60 years of age, of at least 70 years of age, or of at least 80 years of age. In additional embodiments, the subject may suffer from one or more chronic diseases which result in muscle wasting, for example, myopathies, obesity, diabetes, cancer, COPD, cachexia, bacterial infections, and viral infections, or undergoing a therapy for a disease such as chemotherapy, or steroid or statin treatment. In yet additional embodiments, the subject may be physically immobilized, for example, by hospitalization, a cast, bed rest, or with sprained or broken limbs. In more specific embodiments, the subject is a human subject.

[00028] Any suitable source of HMB may be employed in the methods and nutritional compositions of the invention, including the free acid, a salt, including hydrated or anhydrous salts, an ester, a lactone, or other product form that otherwise provides a bioavailable form of HMB when administered. Non-limiting examples of suitable salts of HMB for use herein include HMB salts, hydrated or anhydrous, of sodium, potassium, magnesium, chromium, or calcium, or other non-toxic salt form. In a more specific embodiment, the HMB is in the form of calcium HMB, or, more specifically, calcium HMB monohydrate.

[00029] The methods and nutritional compositions as described herein employ an amount of HMB that is effective, in combination with the citrus flavonoid, to promote muscle regeneration, and, more specifically, to promote muscle regeneration to an extent greater than that achieved with HMB alone. In specific embodiments of the methods of the invention, HMB is administered to the subject in an amount of about 0.1 to about 10 g/day, about 0.1 to about 5 g/day, about 0.5 to about 5 g/day, about 0.5 to about 3 g/day, or about 0.5 to about 1.5 g/day.

[00030] Various citrus flavonoids are suitable for use in the present methods and nutritional compositions. Examples include, but are not limited to, hesperidin, hesperetin, which is the aglycone of hesperidin, narirutin, diosmin, isonaringin, naringin, and didymin, which may be used alone or in combinations of any two or more. In a specific embodiment, the citrus flavonoid comprises hesperidin, hesperetin, or a combination thereof. Hesperidin is the major flavonoid present in sweet oranges, but is also found in other citrus fruits including lemon, lime, grapefruit, mandarin and other classes of oranges. Upon ingestion, hesperidin is hydrolyzed into hesperetin (the aglycone) by colonic microbiota prior to its absorption. The citrus flavonoid may be administered as a citrus extract, or as a purified natural or synthetically produced ingredient, or as a mixture of such ingredients where more than one citrus flavonoid is employed. The methods and nutritional compositions as described herein employ an amount of citrus flavonoid that is effective, in combination with the HMB, to promote muscle regeneration, and, more specifically, to promote muscle regeneration to an extent greater than that achieved with the citrus flavonoid alone. In specific embodiments of the methods of the invention, citrus flavonoid is administered in an amount of about 50 to about 1000 mg/day, about 50 to about 800 mg/day, about 50 to about 500 mg/day, about 50 to about 300 mg/day, about 100 to about 300 mg/day, or about 100 to about 500 mg/day.

[00031] In another specific embodiment of the inventive methods, HMB and at least one citrus flavonoid are administered to the subject daily for a period of time. In specific embodiments, the HMB and at least one citrus flavonoid are administered to the subject one, two or three or more times per day, for a period of 3 days, 5 days, 7 days, 10 days, two weeks, one month, or more. In a more specific embodiment, HMB and at least one citrus flavonoid are administered to the subject at least once per day for at least 3 days, for at least 5 days, for at least 7 days, for at least 14 days, or for at least 30 days.

[00032] The HMB and at least one citrus flavonoid may be administered simultaneously or sequentially. In a specific embodiment, the HMB and at least one citrus flavonoid are administered to the subject simultaneously. In a more specific embodiment, the HMB and at least one citrus flavonoid are administered to the subject simultaneously in a nutritional composition. In a specific embodiment, the HMB and at least one citrus flavonoid are administered to the subject simultaneously in a nutritional composition comprising protein, fat and carbohydrate. [00033] The nutritional compositions employed in specific embodiments of the invention and comprising protein, fat, carbohydrate, HMB, and at least one citrus flavonoid may be in the form of a liquid nutritional composition or a powdered nutritional composition. The term “liquid nutritional composition” as used herein, unless otherwise specified, encompasses all forms of liquid nutritional compositions, including emulsified liquids, concentrated liquids intended for dilution, for example, by addition of water, ready-to-drink liquids, and liquids that are reconstituted from powdered form by addition of liquid, for example, by addition of water or juice. The nutritional compositions are suitable for consumption by a human and, in a specific embodiment, are in a form suitable for oral consumption.

[00034] The nutritional compositions comprise an amount of HMB that is effective, in combination with the citrus flavonoid, to promote muscle regeneration. In specific embodiments the nutritional compositions comprise HMB in an amount effective, in combination with the citrus flavonoid, to promote muscle regeneration to an extent greater than that achieved with HMB alone. In additional specific embodiments, the nutritional compositions comprise HMB in an amount of about 0.1 to about 10 g per 237 ml serving (an 8 ounce serving), about 0.1 to about 5 g per 237 ml serving, about 0.5 to about 5 g per 237 ml serving, about 0.5 to about 3 g per 237 ml serving, or about 0.5 to about 1.5 g per 237 ml serving. The nutritional compositions comprise an amount of citrus flavonoid that is effective, in combination with the HMB, to promote muscle regeneration. In specific embodiments the nutritional compositions comprise citrus flavonoid in an amount effective, in combination with the HMB, to promote muscle regeneration to an extent greater than that achieved with citrus flavonoid alone. In additional specific embodiments, the nutritional compositions comprise citrus flavonoid in an amount of about 50 to about 1000 mg per 237 ml serving, about 50 to about 800 mg per 237 ml serving, about 50 to about 500 mg per 237 ml serving, about 50 to about 300 mg per 237 ml serving, about 100 to about 300 mg per 237 ml serving, or about 100 to about 500 mg per 237 ml serving. [00035] The protein which is contained in the nutritional composition may be any one or more proteins known for use in nutritional compositions. A wide variety of sources and types of protein can be used in the nutritional compositions. For example, the source of protein may include, but is not limited to, intact, hydrolyzed, and partially hydrolyzed protein, which may be derived from any suitable source such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, brown rice, oats, barley, etc.), vegetable (e.g., soy, corn, pea, yellow pea, fava bean, chickpea, canola, potato, mung, ancient grains such as quinoa, amaranth, and chia, hemp, flax seed, etc.), nuts (e.g., almond, cashew, etc.), and combinations of two or more thereof. The protein may also include one or a mixture of naturally occurring or synthetic amino acids (often described as free amino acids) and/or their metabolites, known for use in nutritional products, alone or in combination with the intact, hydrolyzed, and/or partially hydrolyzed proteins described herein.

[00036] More specific examples of sources of protein which are suitable for use in the exemplary nutritional compositions described herein include, but are not limited to, whole egg powder, egg yolk powder, egg white powder, whey protein (including, but not limited to, whey protein components such as a-lactalbumin and/or p-lactoglobulin), whey protein concentrate, whey protein isolate, whey protein hydrolysate, acid casein, casein protein isolate, sodium caseinate, calcium caseinate potassium caseinate, casein hydrolysate, milk protein concentrate, milk protein isolate, milk protein hydrolysate, nonfat dry milk, condensed skim milk, whole cow’s milk, partially or completely defatted milk, coconut milk, soy protein concentrate, soy protein isolate, soy protein hydrolysate, pea protein concentrate, pea protein isolate, pea protein hydrolysate, rice protein concentrate, rice protein isolate, rice protein hydrolysate, barley rice protein, fava bean protein concentrate, fava bean protein isolate, fava bean protein hydrolysate, collagen protein, collagen protein isolate, meat protein such as beef protein isolate and/or chicken protein isolate, potato protein, chickpea protein, canola protein, mung protein, quinoa protein, amaranth protein, chia protein, hemp protein, flax seed protein, almond protein, cashew protein, earthworm protein, insect protein, and combinations of two or more thereof. The nutritional compositions can include any individual source of protein or a combination of any two or more sources of protein. In specific embodiments, the nutritional compositions comprise at least one milk protein, or at least one plant protein, or at least one milk protein and at least one plant protein.

[00037] The amount of the source of protein in the nutritional compositions can vary considerably depending upon, for example, the specific dietary needs of the subject and/or the form of the composition, i.e., liquid or powder. For example, in specific embodiments, the source of protein comprises from about 1 wt% to about 30 wt% of the nutritional composition. In more specific embodiments, the source of protein comprises from about 2 wt% to about 25 wt% of the nutritional composition, including about 2 wt% to about 20 wt%, about 2 wt% to about 15 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 25 wt%, about 10 wt% to about 25 wt%, or about 5 wt% to about 15 wt% of the nutritional composition.

[00038] The nutritional compositions also include fat. The term “fat” as used herein, unless otherwise specified, refers to lipids, fats, oils, and combinations thereof. Sources of fat suitable for use in the nutritional composition include, but are not limited to, algal oil, canola oil, flaxseed oil, borage oil, safflower oil, high oleic safflower oil, high gamma-linolenic acid (GLA) safflower oil, corn oil, soy oil, sunflower oil, high oleic sunflower oil, cottonseed oil, coconut oil, fractionated coconut oil, medium chain triglycerides (MCT) oil, palm oil, palm kernel oil, palm olein, lecithin, and long chain polyunsaturated fatty acids such as docosahexanoic acid (DHA), arachidonic acid (ARA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), and combinations thereof. The nutritional compositions can include any individual source of fat or a combination of two or more sources of fat.

[00039] In specific embodiments, the nutritional compositions comprise about 0.5 wt% to about 20 wt% of a source of fat. In more specific embodiments, the source of fat comprises about 0.5 wt% to about 18 wt% of the nutritional composition, including about 0.5 wt% to about 15 wt%, about 0.5 wt% to about 10 wt%, about 0.5 wt% to about 5 wt%, about 2 wt% to about 8 wt%, about 2 wt% to about 10 wt%, about 5 wt% to about 15 wt%, or about 5 wt% to about 20 wt% of the nutritional composition.

[00040] Sources of carbohydrates suitable for use in the nutritional compositions may be simple or complex, or variations, or combinations thereof. Various sources of carbohydrate may be used so long as the source is suitable for use in a nutritional composition and is otherwise compatible with any other selected ingredients or features present in the nutritional composition. Non-limiting examples of sources of carbohydrates suitable for use in the nutritional compositions include maltodextrin, hydrolyzed or modified starch, hydrolyzed or modified cornstarch, glucose polymers such as polydextrose and dextrin, corn syrup, corn syrup solids, rice-derived carbohydrates such as rice maltodextrin, brown rice mild powder and brown rice syrup, sucrose, glucose, fructose, lactose, high fructose corn syrup, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), isomaltulose, sucromalt, pullulan, potato starch, corn starch, fructooligosaccharides, galactooligosaccharides, oat fiber, soy fiber, gum arabic, sodium carboxymethylcellulose, methylcellulose, guar gum, gellan gum, locust bean gum, konjac flour, hydroxypropyl methylcellulose, tragacanth gum, karaya gum, gum acacia, chitosan, arabinoglactins, glucomannan, xanthan gum, alginate, pectin, low methoxy pectin, high methoxy pectin, cereal beta-glucans, carrageenan, psyllium, Fibersol™, fruit puree, vegetable puree, isomalto-oligosaccharides, monosaccharides, disaccharides, human milk oligosaccharides (HMOs), tapioca-derived carbohydrates, inulin, other digestion-resistant starches, and artificial sweeteners, and combinations of two or more thereof. The nutritional compositions may include any individual source of carbohydrate or a combination of two or more sources of carbohydrate. [00041] In specific embodiments, a source of carbohydrate is present in an amount from about 5 wt% to about 75 wt% of the nutritional compositions. In more specific embodiments, the source of carbohydrate is present in an amount from about 5 wt% to about 70 wt% of the nutritional composition, including about 5 wt% to about 65 wt%, about 5 wt% to about 50 wt%, about 5 wt% to about 40 wt%, about 5 wt% to about 30 wt%, about 5 wt% to about 25 wt%, about 10 wt% to about 65 wt%, about 20 wt% to about 65 wt%, about 30 wt% to about 65 wt%, about 40 wt% to about 65 wt%, about 40 wt% to about 70 wt%, or about 15 wt% to about 25 wt%, of the nutritional composition. In a specific embodiment, wherein the nutritional composition is a liquid, the source of carbohydrate comprises about 5 wt% to about 30 wt% of the nutritional composition. In more specific liquid embodiments, the carbohydrate comprises about 5 wt% to about 25 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 15 wt%, about 10 wt% to about 25 wt%, about 10 wt% to about 20 wt%, about 15 wt% to about 25 wt%, or about 15 wt% to about 30 wt% of the nutritional composition. In another specific embodiment, wherein the nutritional composition is a powder, the source of carbohydrate comprises about 25 wt% to about 75 wt% of the nutritional composition. In more specific powder embodiments, the carbohydrate comprises about 30 wt% to about 70 wt%, about 35 wt% to about 65 wt%, about 40 wt% to about 65 wt%, about 40 wt% to about 70 wt%, about 50 wt% to about 70 wt%, or about 50 wt% to about 75 wt% of the nutritional composition.

[00042] The relative amounts of the sources of protein, fat and carbohydrate in the nutritional compositions can vary depending upon, for example, the specific dietary needs of the subject and/or the form of the composition, i.e., liquid or powder. In a specific embodiment, the nutritional compositions comprise a source of protein in an amount of about 2 wt % to about 25 wt %, a source of carbohydrate in an amount of about 5 wt % to about 75 wt %, and a source of fat in an amount of about 0.5 wt % to about 20 wt %, based on the weight of the nutritional composition. In a specific embodiment, the nutritional composition is in liquid form and comprises a source of protein in an amount of about 2 wt% to about 25 wt%, a source of carbohydrate in an amount of about 5 wt% to about 30 wt%, and a source of fat in an amount of about 0.5 wt% to about 10 wt%, based on the weight of the nutritional composition. In another specific embodiment, the nutritional composition is in the form of a powder and comprises a source of protein in an amount of about 10 wt% to about 25 wt%, a source of carbohydrate in an amount of about 40 wt% to about 70 wt%, and a source of fat in an amount of about 5 wt% to about 20 wt%, based on the weight of the nutritional composition.

[00043] In specific embodiments, the nutritional composition has a neutral pH, i.e. , a pH of from about 6 to 8 or, more specifically, from about 6 to 7.5. In more specific embodiments, the nutritional composition has a pH of from about 6.5 to 7.2 or, more specifically, from about 6.8 to 7.1.

[00044] The nutritional composition may further comprise one or more additional components that may modify the physical, chemical, aesthetic, or processing characteristics of the nutritional composition or serve as additional nutritional components. Non-limiting examples of additional components include preservatives, emulsifying agents (e.g., lecithin), buffers, sweeteners including artificial sweeteners (e.g., saccharine, aspartame, acesulfame K, sucralose), colorants, flavorants, thickening agents, stabilizers, and so forth.

[00045] Additionally, the nutritional composition may further include vitamins or related nutrients, non-limiting examples of which include vitamin A, vitamin B12, vitamin C, vitamin D, vitamin K, thiamine, riboflavin, pyridoxine, niacin, folic acid, pantothenic acid, biotin, choline, inositol, salts and derivatives thereof, and combinations thereof. Water soluble vitamins may be added in the form of a water-soluble vitamin (WSV) premix and/or oil-soluble vitamins may be added in one or more oil carriers as desired.

[00046] In additional embodiments, the nutritional composition may further include one or more minerals, non-limiting examples of which include calcium, phosphorus, magnesium, iron, zinc, manganese, sodium, potassium, molybdenum, chromium, chloride, and combinations thereof.

[00047] In additional embodiments, the nutritional composition may further include one or more probiotics. The term “probiotic” as used herein refers to a microorganism such as a bacteria or yeast that survives the digestive process to confer a health benefit to the subject. Examples of probiotics that can be included in the nutritional compositions, either alone or in combination, include, but are not limited to, Bifidobacterium (B.), such as B. breve, B. infantis, B. lactis, B. bifidum, B. longum, and B. animalis, and Lactobacillus (/_.), such as L rhamnosus, L. acidophilus, L. fermentum, L. reuteri, Streptococcus thermophilus, Akkermansia, Bacteroides, Enterococcus, Eubacterium, Fecalibacterium, Roseburia, and/or Saccharomyces.

[00048] The nutritional compositions may be formed using any techniques known in the art. In one embodiment, the nutritional compositions may be formed by (a) preparing an aqueous solution comprising protein and carbohydrate; (b) preparing an oil blend comprising fat and oilsoluble components; and (c) mixing together the aqueous solution and the oil blend to form an emulsified liquid nutritional composition. The HMB and citrus flavonoid may be added at any time as desired in the process, for example, to the aqueous solution or to the emulsified blend. The compositions may be spray-dried or otherwise dried, if a powder product is desired. Alternatively, a powder product can be formed by dry blending ingredients. Typically, the nutritional compositions are subjected to a heat treatment which provides sterilization sufficient to maintain microbiological stability of the compositions over a desired shelf-life.

[00049] The improvements provided by the inventive methods are demonstrated in the following Example.

EXAMPLE

[00050] The present example evaluated the effect of HMB, a flavonoid-containing citrus extract (orange peel extract, OPE), hesperidin and hesperetin, each individually, and the effect of combinations of HMB with each of the citrus extract, hesperidin, and hesperetin, respectively, on protein synthesis and on myogenesis.

[00051] As noted, adult skeletal muscle is highly adaptable, responding to the soluble and biophysical cues that it encounters on a daily basis through hypertrophy and hyperplasia. The former relies on the regulation of protein synthesis and degradation rates, while the latter involves the process of myogenesis, which is responsible for regulating the myocyte turnover and supporting rapid muscle regeneration after injury. In order to elicit reparative responses, muscular cells must be activated, increase their numbers (proliferation), and fuse

(differentiation) with the damaged fiber. When fusion of myogenic cells is completed, the size of the newly formed “nascent” myotubes increases. Nascent myotubes need to undergo a maturation process to become fully functional myofibers. Mature muscle fibers are highly specialized and need to acquire a competent excitation-contraction coupling, contractile, and metabolic machineries. Finally, the new muscle tissue is the same as uninjured muscle, not only morphologically but also functionally.

[00052] An in vitro cell culture study was carried out with the objective to evaluate the synergistic effects of HMB and citrus flavonoid on muscle using a well-established rat muscle cell line. Because of their fundamental roles in muscle maintenance and adaptation, myoblasts and myotubes are frequently studied as in vitro models of myogenesis (differentiation phase) and newly formed myotubes (maturation and hypertrophy) to evaluate the effect of different ingredients on muscle regeneration capacity. The L6.C11 rat skeletal muscle myoblast line (European Collection of Cell Cultures no. 92102119) was employed in this study.

[00053] First, the expression of creatine kinase, which is involved in and a measure of cell differentiation, was studied. Specifically, the L6.C11 rat skeletal muscle myoblast cells were incubated for 30 min in the presence of HMB, OPE, hesperidin and hesperetin, each individually, and in the presence of combinations of HMB with each of OPE, hesperidin, and hesperetin, respectively. Cells were also incubated without any additive to serve as a control. Following this treatment, plates were processed as described by Giron et al, Diabetologia, 51:1285 (2008). The protein concentration of the supernatants was measured using a bicinchoninic acid method (Bio-Rad, Madrid, Spain). Proteins (40 pg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, and immunoblotted with specific antibodies; the immunoblots were developed by an enhanced chemiluminescence detection method. Antibodies against non-phosphorylated kinase (GAPDH) was used as load control. [00054] The effects of the different ingredients on the L6.C11 rat skeletal muscle myoblast cell differentiation, using creatine kinase (CK) as a biochemical marker, are shown in Figs. 1-3. Data were normalized to the control and presented as media ± SEM (n=6). Differences between groups were evaluated by unpaired Student’s t-test. Statistical significance was defined by p- values < 0.05, with a, b, c and d in Figs. 1-3 indicating significant differences. Statistical analysis was carried out with GraphPad Prism 8.0.2.

[00055] Figs. 1-3 show that cell incubation with each of HMB, OPE, hesperidin, and hesperetin provided significant increases in muscle cell differentiation as measured by CK expression and compared with the control. Fig. 1 shows the combination of HMB and OPE did not show a significant increase as compared with OPE alone, while Figs. 2 and 3 show that, surprisingly, the combination of HMB with hesperidin and hesperetin, respectively, synergistically increased the CK expression as compared with the control and as compared with each component individually. A comparison of Figs. 2 and 3 with Fig. 1 may indicate that amount of citrus flavonoid in OPE was not sufficient, in combination with HMB, to provide the synergistic effect exhibited by the combinations of HMB and hesperidin, and HMB and hesperetin.

[00056] Next, the L6.C11 rat skeletal muscle myoblast line was grown in Dulbecco’s Modified Eagle’s Medium (DM EM) supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 units/mL penicillin, and 0.1 mg/mL streptomycin and was maintained at subconfluent densities. When the myoblasts reached about 80% confluency, they were differentiated into myotubes by exchanging the growth medium with a differentiation medium comprising DMEM supplemented with 2% (v/v) FCS for 5-6 days.

[00057] After differentiation, newly formed myotubes were incubated with HMB (12.5 pM) and with the different citrus flavonoid sources: citrus flavanol extract (OPE, 25 pg/mL), hesperidin (10 pM), or hesperetin (1 pM), each individually, and each in combination with HMB. Myotubes were also incubated without any additive to serve as a control. [00058] Protein synthesis was measured as a marker of myotubes maturation/ hypertrophy according to the method described by Gulve et al, Biochemistry Journal, 2Q0.377 (1989) with the following modifications: the L6.C11 cells were plated on 48-well tissue culture plates, differentiated for 5 days, and then starve treated with the different effectors for 2 h in media with 10% FCS and 0.8 mM tyrosine. Cells were then spiked with 1 pCi/mL of L-[ring-3,5-3H]-tyrosine and incubated for 1 h. The reaction was stopped by placing the plates on ice. Wells were thoroughly washed two times with ice-cold phosphate-buffered saline media containing 2 mM non-radioactive tyrosine, and cells were then lysed in 0.1 mM NaOH/0.1% sodium deoxycholate. Proteins were precipitated by adding cold 20% tricarboxylic acid cycle (Sigma). This mixture was then incubated at 4°C for 15 min. Following centrifugation (16,000 x g for 10 min), the pellet was thoroughly washed with a cold 10% tricarboxylic acid cycle, and then precipitated proteins were dissolved in 0.1 mL of 1M NaOH. An aliquot (5 pL) of the NaOH- solubilized material was used for total protein quantification, and the remaining dissolved proteins were neutralized with 1M HCI and mixed with Ready Safe scintillation fluid (Beckman Coulter, Brea, CA). The radiolabel was determined with a scintillation counter (Beckman Coulter). Data were computed as disintegrations per minute per microgram of proteins and expressed as relative units (%).

[00059] The results are set forth in Figs. 4-6. Data were normalized to the control and presented as media ± SEM (n=6). Differences between groups were evaluated by unpaired Student’s t-test. Statistical significance was defined by p-values < 0.05, with a, b and c in Figs. 4-6 indicating significant differences. Statistical analysis was carried out with GraphPad Prism 8.0.2.

[00060] Fig. 4 shows the effect of HMB, OPE, and a combination of HMB and OPE on protein synthesis. While HMB alone provided an increase in protein synthesis (about 23% as compared with the control), OPE alone did not provide any significant effect on protein synthesis. On the other hand, the combination of HMB and OPE surprisingly increased protein synthesis by about 45% as compared with the control. Fig. 5 shows that while hesperidin alone provided a modest increase in protein synthesis (about 16% as compared with the control), the combination of HMB and hesperidin surprisingly increased protein synthesis by about 48% as compared with the control. Finally, Fig. 6 shows that hesperetin alone did not provide any significant effect on protein synthesis, but the combination of HMB and hesperetin surprisingly increased protein synthesis by about 43% as compared with the control.

[00061]

[00062] These results indicate that combinations of citrus flavonoid and HMB positively and synergistically affect muscle protein synthesis and differentiation, and therefore evidence that the combinations promote muscle regeneration.

[00063] While the present application has been illustrated by the description of embodiments thereof, and while the embodiments have been described in considerable detail, such descriptions are not intended to restrict or in any way limit the scope of the appended claims to such detail. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention, in its broader aspects, is not limited to the specific details, the representative methods or compositions, or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the general inventive concept.