Glyceollins as exercise mimetic

Abstract

Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance, and it is effective for the prevention and treatment of obesity and metabolic syndrome. Previously, it was shown that agonists for estrogen receptor-related receptors (ERRs), an orphan nuclear receptor family, and agonists for REV-ERB α / β, another nuclear receptor family, improved lipid oxidation in mitochondria and skeletal muscle exercise capacity. Here, it is demonstrated that glyceollin I, glyceollin II, and glyceollin III, natural products extracted from soybeans, enhanced ERRs' transcription activator activity, especially ERRα and γ receptors, and that combination of all three types of glyceollins enhanced REV-ERB α / β's transcription repressor activity. These results indicate the feasibility of individual glyceollin I, II, III, and a mixture of all three glyceollins as exercise mimetics that may be effective in the treatment of numerous obesity related diseases and to improve muscle function in the aging.

Description

[0001] Attorney Docket No.:10457-582PC0

[0002] Glyceollins as Exercise Mimetic

[0003] GOVERNMENT SUPPORT CLAUSE

[0004] This invention was made with government support under AG077160 and AG060769 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] 1. Field of the Invention

[0007] The invention relates to a pharmaceutical composition for enhancing muscle functionality in physical exercise, and particularly to a composition comprising glyceollins I, II. and III.

[0008] 2. Background

[0009] According to the definition of Johns Hopkins Medicine, metabolic syndrome refers to “the presence of a cluster of risk factors specific for cardiovascular disease”, which “greatly raises the risk of developing diabetes, heart disease, stroke, or all three” (https: / / www.hopkinsmedicine.org). Obesity, which is defined as “a state of excess adipose tissue mass that adversely affects health” (Harrison's Principles of Internal Medicine, 21e. McGraw Hill LLC), is one of the factors involved with metabolic syndrome, which is manifested by high blood pressure, high fasting blood glucose level, high triglyceride levels and low HDL (good) cholesterol. Since obesity makes it more difficult for the body to respond to insulin, which is attributed to the overabundance of circulating fatty acids, the blood sugar level increases, leading to type 2 diabetes.

[0010] As a substantial contributor to the development and progression of obesity, lack of physical exercise can be considered, which also increases the risk of various diseases in addition to metabolic syndrome, including insulin resistance, type 2 diabetes, hyperlipidemia of high triglycerides and high cholesterol, atherosclerosis, metabolic associated steatohepatitis (MASH), as well as sarcopenia, osteoporosis, the aging process of muscle, and certain cancers. Physical exercise is also known to improve cognition and to slow dementia. Therefore, exercise is an effective treatment for such diseases, and even a single bout of exercise improves whole-bodyAttorney Docket No.:10457-582PC0

[0011] insulin sensitivity for up to 48 h after exercise cessation (Koopman et al., 2004) and increase basal energy expenditure beyond the point of exercise termination (Bprsheim et al., 2003).

[0012] Therefore, exercise can be a more effective treatment than currently available pharmacological therapies, when combined with dietary regimen.

[0013] Physical exercise is generally classified as either aerobic exercise (endurance-based high-frequency repetition with relatively low load) and anaerobic exercise (resistance and strengthbased low-frequency repetition with relatively high load). Aerobic exercise is fueled by oxygen from breathing while repeating low load movement for at least 30 minutes or longer. Anaerobic exercise is high-intensity exercise that’s meant to be short and fast, and during exercise, the body uses the energy that’s already stored in muscles.

[0014] The skeletal muscle is one of the primary tissues that adapt to exercise in order to physically and metabolically acclimatize to the increase in utilization. Skeletal muscles use both glucose and fatty acids as their main source of energy. In general, glucose is the fuel preferentially used for short- and high-load exercise (anaerobic exercise), and as the duration of energy demand lengthens (long- and low load exercise), skeletal muscle switches its energy source to fatty acids (aerobic exercise) (Meex et al., 2010). Increased fatty acid utilization decreases fat storage in the tissues and improves overall metabolism functionality, and it is generally associated with improved glucose tolerance and insulin sensitivity (Donnelly et al., 2013).

[0015] In addition, as for a long-term adaptation, physical exercise triggers dramatic changes in skeletal muscle genes and protein expression to improve muscle function (strength and endurance), which can be detected after single bouts of exercise (acute exercise) and repeated bouts of exercise (training) (Egan et al., 2013). Both aerobic and anaerobic / resistance exercise are effective in preventing and treating obesity and various metabolic syndrome related diseases, and induce distinct physiological adaptations within the skeletal muscle.

[0016] SUMMARY

[0017] A key adaptation of skeletal muscle in response to aerobic exercise is to increase oxidative metabolic capacity mediated by enhanced mitochondrial respiratory capacity, which allows for more efficient energy production and enhanced exercise endurance (Meex et al., 2010) and consequently reduces obesity.Attorney Docket No.:10457-582PC0

[0018] Interestingly, high energy demand tissues, such as muscle, heart, and liver display high levels of estrogen receptor-related receptors (ERRs) expression (Giguere et al., 1988; Sladek et al., 1997; Chen et al., 1999). ERRs (ERRa, ERR , and ERRy) are orphan members of the nuclear receptor superfamily. ERRa is expressed mainly in brown adipose tissue, intestine, and skeletal muscles, and functions as a sensor of energy metabolism. ERRp is expressed at low levels in adult skeletal muscle, heart, kidney, eyes, and ears. This isoform is known to play an important role in early development, and its postnatal expression is highly restricted. ERRy is expressed mainly in skeletal muscle, heart, pancreas, kidney, placenta, spinal cord and brain (Eudy et al., 1998; Heard etal., 2000; Hong et al., 1999).

[0019] ERRs are constitutively active receptors, which are most closely related to estrogen receptors (ERs) in terms of sequence homology but do not display the estrogen-binding properties of the ERs (Giguere et al., 1988). A range of target genes whose transcription is activated by ERRs have been identified, which includes enzymes and regulatory proteins in energy production pathways involved in fatty acid oxidation, the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, and mitochondrial biogenesis (Audet- Walsh and Giguere, 2015; Fan and Evans, 2015).

[0020] Previously, it was demonstrated by this lab that a synthetic agonist, SLU-PP-332, which activates ERR receptors, improved exercise performance, enhanced cognition function, and have utility in treatment of heart failure (Billon et al., 2024; Billon et al., 2023). Here, it is discovered that natural products (derived from soybeans) glyceollin I, glyceollin II, and glyceollin III function as ERR agonists.

[0021] Glyceollins are phytoalexins produced by soybeans under stress (Aspergillus oryzae -filamentous fungus used in soybean fermentation in food production). Phytoalexins refer to polyphenolic compounds produced by plants as defense molecules against phytopathogens. Glyceollins are produced in very small amounts in healthy plants, but their synthesis is stimulated by infectious agents and other stresses (high temperature, UV radiation, etc.).

[0022] The REV-ERB s represent another nuclear receptor subfamily, which are involved in energy metabolism whose activation is associated with exercise mimetic activity (Woldt et al., 2013). The REV-ERB s acquired their unusual name owing to the unique genomic organization of NR1D1, which encodes REV-ERBa. REV-ERBa is encoded by the opposite DNA strand of the ERBAAttorney Docket No.:10457-582PC0

[0023] (also known as THR A) oncogene (Lazar etal., 1989; Miyajimaet < / ., 1989; Miyajim et^ / ., 1988), which encodes the thyroid hormone receptor-a, wherein the thyroid hormone is well known for controlling metabolism, growth, and many other bodily functions. Thus, the name REV-ERB is derived from ‘reverse strand of ERBA’. Both REV-ERBa and the closely related REV-ERBp (encoded by NR1D2) have an atypical LBD (ligand-binding domain) that lacks the carboxyterminal activation function 2 (AF2) region (Dumas et al., 1994; Forman et al., 1994; Bonnelye et al., 1994) (FIG. 4). Because the AF2 region recognizes co-activators that are necessary for transcriptional activation, REV-ERBa and REV-ERBp are generally characterized as being unable to activate transcription. Indeed, the REV- ERB s are constitutive repressors of transcription owing to their constant binding of co-repressors such as the nuclear receptor co-repressor 1 (NCoRl) (Yin and Lazar, 2005).

[0024] It was previously demonstrated by this lab that a synthetic REV-ERB ligand, SR9009, functioned as exercise mimetics, and also improved the metabolic profile in mouse models of obesity and metabolic disease (Woldt et al., 2013; Kojetin and Burris, 2014). Here, it was discovered that a combination of glyceollin I, glyceollin II, and glyceollin III function effectively as a REV-ERB ligand complex, enhancing its transcription repressor activity. These individual compounds showed enhancing transcription repressor activity of REV-ERB in cell based luciferase assays (mix is best though) whereas in the biochemical radioligand binding assay in the presence of STL 1267, a new type of synthetic REV-ERB agonist, there seems to be much higher preference for the mix vs. individual compounds, providing a unique observation that the glyceollin mixture may be used to provide medicinal benefits.

[0025] Therefore, the main objective of this disclosure is to develop a pharmaceutical composition comprising glyceollins, in particular the combination of all three glyceollins, with or without other ingredients such as vitamins, minerals, essential amino acids, phytochemicals, pharmaceutical excipients or food additives, for use as an exercise mimetic. The glyceollin compositions can be formulated into dietary supplements, powder / granules with plant herbs for tea or with protein powder for shake blend, or liquid for an energy drink. Such glyceollin products will have multiple effects, e.g., increasing oxidative metabolism in skeletal muscle, skeletal muscle fiber type switch (to oxidative fibers), enhancing exercise endurance, and reducing obesity and symptoms of various obesity-related diseases such as metabolic syndrome, insulin resistance, type 2 diabetes,Attorney Docket No.:10457-582PC0

[0026] hyperlipidemia of high triglycerides and high cholesterol, atherosclerosis and high blood pressure, metabolic associated steatohepatitis (MASH), sarcopenia, osteoporosis, the aging process of muscle and cognition, dementia, and certain cancers.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1. The structures of isoflavonoids and glyceollins (Yue et al., 2023)

[0029] Figure 2. De Novo Synthesis of glyceollins (Luniwal et al., 2011)

[0030] Figure 3. (a) Purification of glyceollins from stressed soybeans (Abe et al., 2021). (b) Table 1. Amount of isoflavones and their metabolites in soybean seeds inoculated with or without Aspergillus oryzae.

[0031] Figure 4. (a) The general organizational structure of members of the nuclear receptor superfamily, (b) Structural organization of the REV-ERB s. (c) Structural organization of ERRs and comparison to the estrogen receptors (ERa and ER|3). Numbers above each receptor represent the amino acid position. Percentages indicate amino acid identity within a particular domain relative to either REV-ERBa. A / B, C, D, E and F refer to classically defined regions in the nuclear receptor domain structure. DBD, DNA-binding domain; LBD, ligand-binding domain.

[0032] Figure 5. Glyceollin I, II, and III activate transcriptional activity of ERRa and ERRy in the presence of ERR response element sequences (three) in phosphoglycerate kinase (PGK) promoter in nanoluciferase reporter plasmid construct, (a) Illustration of promoterreporter (nanoluciferase) constructs on PGK promoter for the measurement of transcription activation in the presence of glyceollin. Glyceollins were also assessed for activity on an identical reporter plasmid construct that did not contain ERRE DNA response elements (OXERRE PGK: :nluc) to eliminate any potential non-specific (not ERR mediated) effects of glyceollins on luciferase expression or activity, (b) ERRa and ERRy cotransfection assay in the presence of glyceollin I, II, or III. Data are reported as specific to ERRa or ERRy based on cotransfection of HEK293 cells with either human ERRa or ERRy expression vectors with either the reporter vector containing ERREs or no ERREs to provide the ability to determine ERR specific activity, (c) Example of activity of known ERR activators, SLU-PP-332 and SLU-PP-915, in the ERRa and ERRy cotransfection assays.Attorney Docket No.:10457-582PC0

[0033] Figure 6. A mixture of glyceollin T, TI, and TIT reduced STL1267 binding to REV-ERB. (a) REVERB a radioligand displacement binding assay with increasing concentrations of glyceollin, I, IT, ITT and mixture of all three glyceollins. (b) (a) REV-ERB 0 radioligand displacement binding assay with increasing concentrations of glyceollin, I, IT, ITT and mixture of all three glyceollins.

[0034] Figure 7. Glyceollin I, IT, and ITT activate transcriptional repressor activity of REV-ERB in multiple REV- ERB - responsive reporter assays, (a) Illustration of promoter: reporter (luciferase) constructs on Bmall, Cyp8bl, and SlclOal promoters (containing natural REV-ERB DNA response elements) for transcription repression in the presence of glyceollin. REV-ERB a cotransfection assay using (b) Bmall, (c) Cyp8bl, and (d) SlclOal promoter:reporter constructs. Figure 8. Cell-based cotransfection assay for REV-ERBa with increasing concentrations of glyceollin I and SR9009, an agonist for REV-ERB.

[0035] Figure 9. Glyceollin administration increases exercise endurance in mice. Male C57B16 mice 8-10 weeks old (N= 15- 16 / group) were administered vehicle (PBS / Tween 80 / Cremophor EL / DMSO (80% / 5% / 5% / 10%)) or 20 mg / kg glyceollin once per day for 30 days subcutaneously. Glyceollin administration results in a 22% increase endurance. **, p<0.01 vs. vehicle via Student’s t test (2-tailed)

[0036] Figure 10: Glyceollin induces mitochondrial respiratory gene expression. Expression of mitochondrial complex I genes (ND1-ND6) and the nuclear-encoded respiratory gene Ndufvl was quantified by qPCR and expressed relative to vehicle controls (Vehicle = 100%). Bars represent mean ± SEM. *p < 0.05 vs vehicle (one-tailed test). Male C57B16 mice 8-10 weeks old (N=15-l 6 / group) were administered vehicle (PBS / Tween 80 / Cremophor EL / DMSO (80% / 5% / 5% / 10%)) or 20 mg / kg glyceollin once per day for 30 days subcutaneously. Skeletal muscle (gastrocnemius) gene expression was then assessed by QPCR. ND1-ND6 =mtNDl through mtND6 which are mitochondrially encoded NADHmbiquinone oxidoreductase core subunit (1-6). Ndufvl is the nuclear encoded mitochondrial gene NADHmbiquinone oxidoreductase core subunit V 1.

[0037] DETAILED DESCRIPTION

[0038] A. DefinitionsAttorney Docket No.:10457-582PC0

[0039] Embodiments of materials and methods are described herein; any methods and materials similar or equivalent to those described herein can be used in the practice of or testing of the invention.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to be limiting. Also, the entire teachings of any patents, patent applications or other publications referred to herein are incorporated by reference herein as if fully set forth herein.

[0041] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, pathology, physiology, genetics, protein, and nucleic acid chemistry described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed through the present specification unless otherwise indicated.

[0042] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / - 10% or less, +1-5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0043] All ranges recited herein include the endpoints, including those that recite a range “between” two values. Terms such as “about,” “generally,” “substantially,” “approximately” and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.Attorney Docket No.:10457-582PC0

[0044] Unless otherwise indicated, as used herein, “a” and “an” include the plural, such that, e.g., “a compound” can mean at least one compound, as well as a plurality of compounds, i.e., more than one compound. As understood by one of skill in the art. the term “compound” can be used to refer to a single compound or more than one compound.

[0045] The present invention can “comprise” (open ended) the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0046] Where used herein, the term “and / or” when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition of the instant invention is described as containing characteristics A, B, and / or C, the composition can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0047] As used herein, the term "subject" refers to any mammals, and in particular humans, who are suspected of having, has been diagnosed as having, or is at risk of developing obesity, metabolic syndrome, insulin resistance, type 2 diabetes, hyperlipidemia of high triglycerides and high cholesterol, atherosclerosis, MASH, sarcopenia, osteoporosis, dementia, and certain cancers, which can be ameliorated, treated, or prevented by glyceollins according to the embodiments of the invention.

[0048] As used herein, the term "treatment" or “treat” in the context of pharmacological or clinical meaning refers to intervention of disease, disorder, condition or one or more symptoms thereof to obtain a desired patho-physiological effect, such as: preventing or inhibiting the development of the disease, disorder, condition, or one or more symptoms thereof; slowing or delaying the progress of the disease, disorder, condition, or one or more symptoms thereof; stabilizing (i.e., not worsening) a state of the disease, disorder, condition, or one or more symptoms thereof; and relieving, palliating, alleviating, or ameliorating the severity of the disease, disorder, condition, or one or more symptoms thereof; or preventing or inhibiting remission, whether partial or total andAttorney Docket No.:10457-582PC0

[0049] whether detectable or undetectable. “Treatment,” or “treat” may not necessarily indicate complete eradication or cure of the disease, disorder, condition, or associated symptoms thereof.

[0050] As used herein, the term "administering" refers to introducing the inventive composition to a subject through various routes, and can be performed using any of the various methods for drug delivery known to those skilled in the art. Modes of drug administration include, but are not limited to enteral (including oral, sublingual / buccal, and rectal administration), parenteral (including subcutaneous (SC / SQ), intravenous (IV), intradermal (ID), intramuscular (IM), intraperitoneal (IP), intraarterial, intracardiac, intraarticular, and intrathecal injection or infusion), and topical, transdermal, and mucosal (including inhalation and nasal administration) administration to deliver a therapeutically effective amount of the drug to the cells or tissue to which it is targeted. Preferably, administration according to the embodiments of this invention is oral administration, but if need be, parenteral or transdermal administration as well as pellet implant can be contemplated.

[0051] As used herein, a "therapeutically effective amount” or an “effective amount” refers to a quantity of a drug, at dosages and for periods of time necessary, that can achieve a desired effect. It can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the drug to generate a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of a drug are outweighed by the therapeutically beneficial effects. The “effective amount” needs to be determined considering the pharmacodynamics and pharmacokinetics of the drug to achieve the intended result.

[0052] As used herein, the term “composition” refers to a pharmaceutical composition, meaning a mixture of compounds or substances suitable for administering to a subject, which includes one or more pharmaceutically active ingredients. For example, a pharmaceutical composition may comprise a certain amount of glyceollin I, II, and / or III as well as necessary pharmaceutical excipients.

[0053] As used herein, the term “pharmaceutical excipient” or “excipient” refers to any substances other than the active ingredient, contained in pharmaceutical dosage forms. The excipients are considered as inert substances, i.e., they do not have any active role in therapeutics, but they canAttorney Docket No.:10457-582PC0

[0054] be used to support the process to produce an effective product. Examples of excipients can be found in Remington: The Science and Practice of Pharmacy, 23rdEd.

[0055] As used herein, the term “dosage form” refers to a pharmaceutical preparation in which a specific mixture of active ingredients and inactive components (excipients) are formulated in a particular shape or form to facilitated administration and accurate delivery of active ingredients, and / or to be presented in the market. Solid dosage forms include powders, granules, capsules, tablets, pills, dragees (or dragees), modified-release forms of tablets, pills, or capsules (extended / sustained release), cachets, lozenges (or troches), chewable tablets, orally disintegrating tablets (ODTs), gummies, films, and suppositories. Semi-solid dosage forms include ointments (greasy, hydrocarbon-based), creams (water-in-oil / oil-in-water emulsions), gels (non-greasy, aqueous), pastes (high powder content), lotions, jellies, and poultices. Liquid dosage forms include solutions (e.g., oral, topical, or ophthalmic solutions), syrups (i.e., viscous oral liquids containing high concentrations of sucrose or sugars), suspensions (i.e., liquid preparations with solid particles dispersed in a liquid base, requiring shaking before use), elixirs (i.e., clear, sweetened, hydro-alcoholic liquid preparations for oral use), emulsions (i.e., dispersion of two immiscible liquids (oil and water) stabilized by an emulsifying agent), drops (i.e., concentrated liquid preparations meant to be taken in small amounts, often with a dropper), lotions (i.e., topical liquid preparations applied to the skin without friction, liniments (i.e., topical preparations applied with friction), injections (e.g., solutions, suspensions, or emulsions to be administered via syringe), gargles / mouthwashes, and enemas (e.g., solution, emulsion, or suspension). Gaseous dosage forms include inhalation aerosols, inhalations, nasal aerosols, sprays and foams, and medical gases.

[0056] As used herein, “dietary supplement” refers to a substance that can help improve or maintain overall health, and / or meet daily requirements of essential nutrients. Generally, the product is intended to treat (relieve or ameliorate) or prevent diseases. Dietary supplements are manufactured in many forms for oral usage, including tablets, pills, capsules, soft gels, gummies, powders, granules, bars, and liquids. The products can be added to food or taken with conventional food or alone between meals. The inventive dietary supplements may include, in addition to glyceollins, vitamins (including vitamin A, Bi (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), Be (pyridoxine), B7 (biotin), B9 (folate / folic acid), B12 (cobalamin), C (ascorbic acid), D, E (tocopherol), and K; minerals (including calcium, chloride, chromium, copper, iodine,Attorney Docket No.:10457-582PC0

[0057] iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, vanadium, zinc); essential amino acids (including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine); botanical herbs (extracts from plants or plant parts, including acai berry, alfalfa, aloe vera, ashwagandha, astragalus, Bacopa monnieri, bee pollen, berberine, bilberry, bitter orange, black cohosh, blackcurrant, blueberry, bromelain, butterbur, capsaicinoids. cat’s claw, chamomile, chasteberry, cherry, cinnamon, coffee bean, coffee fruit, cranberry, curcumin, dandelion, date, deer velvet, echinacea, elderberry, evening primrose oil, fenugreek, feverfew, flaxseed, Garcinia cambogia, garlic, ginger, ginger root, ginkgo, ginseng, goji berry, goldenseal, grape seed extract, green tea, guar gum, hawthorn, hoodia, horse chestnut, juniper berry, kava, lemon, lavender, licorice root, matcha, milk thistle, mistletoe, mushrooms, noni, passionflower, peppermint oil, pomegranate, raspberry, red clover, red date, rhodiola. sage, saw palmetto, soy, St. John’s wort, thunder god vine, turmeric, valerian, white mulberry, white tea, yerba-mate, yohimbe); botanical compounds (including lutein, polyphenols, caffeine, and curcumin); fatty acids (including soybean oil, sunflower seed oil, soy lecithin, sunflower lecithin, fish oil, omega 3, 6, 7, and 9); glucosamine and chondroitin; fibers; and live microbials (commonly referred to as “probiotics”).

[0058] As used herein, “agonist” refers to an activator of a specific receptor, e.g.. nuclear receptor inside the cell or transmembrane receptors on the cell surface, which is a drug or compound that binds to a receptor and activate the receptor to generate the same signal as the natural ligand normally generates when it binds to the receptor.

[0059] As used herein, “metabolic syndrome” refers to a condition with a cluster of risk factors for cardiovascular disease, and it also greatly raises the risk of developing diabetes and / or stroke. According to the National Heart, Lung and Blood Institute (NHLBI), the cluster of risk factors include: abdominal obesity with a waist circumference of more than 35 inches for women and more than 40 inches for men; high blood pressure of over 130 / 80 mm Hg (Normal blood pressure is defined as less than 120 / 80 mm Hg.); high fasting blood glucose levels more than 100 mg / dL; high triglyceride levels more than 150 mg / dL; and low HDL (good) cholesterol less than 40 mg / dL for men and less than 50 mg / dL for women. The cause of metabolic syndrome is not fully understood, but it is generally thought that obesity and a sedentary lifestyle significantly contribute to the onset of metabolic syndrome manifested by symptoms such as high cholesterol, insulinAttorney Docket No.:10457-582PC0

[0060] resistance, and high blood pressure, which may lead to cardiovascular disease and type 2 diabetes. As for other factors for metabolic syndrome, genetic changes in a person's ability to break down fats (lipids) in the blood, older age, and problems in body fat distribution can be attributed. For the treatment of metabolic syndrome, lifestyle management, diet, exercise, medicine, weight-loss surgery can be considered. (https: / / www.hopkinsmedicine.org / health / conditions-and-diseases / metabolic-syndrome)

[0061] As used herein, “food additives” refers to substances primarily added to processed foods, or other foods produced on an industrial scale, for technical purposes, e.g. to improve safety, increase the amount of time a food can be stored, or modify sensory properties of food. Examples of food additives, including glycerol, are well listed in the substance list of the FDA. Non-limiting examples of food additives include: (1) preservatives such as sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, potassium benzoate, calcium benzoate, ethyl p-hydroxybenzoate, sodium ethyl p-hydroxybenzoate, methyl p-hydroxybenzoate, sodium methyl p-hydroxybenzoate, sulphur dioxide, sodium sulphite, sodium hydrogen sulphite, sodium metabisulphite, potassium metabisulphite, calcium sulphite, calcium hydrogen sulphite, potassium hydrogen sulphite, nisin, natamycin, hexamethylene tetramine, dimethyl decarbonate, ethyl lauroyl arginate, potassium nitrite, sodium nitrite, sodium nitrate, potassium nitrate, propionic acid, sodium propionate, calcium propionate, potassium propionate, boric acid, sodium tetraborate; borax, lysozyme; (2) antioxidants such as ascorbic acid, sodium ascorbate, calcium ascorbate, fatty acid esters of ascorbic acid, tocopherols, alpha-tocopherol, gamma-tocopherol, delta-tocopherol, propyl gallate, erythorbic acid, sodium erythorbate, tertiary-butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), extracts of rosemary, 4-hexylresorcinol; (3) sweeteners such as sorbitol, sorbitol syrup, mannitol, acesulfame K, aspartame, cyclamic acid and its Na and Ca salts, isomalt, saccharin and its Na, K and Ca salts, sucralose, thaumatin, neohesperidine DC, steviol glycosides from stevia, enzymatically produced steviol glycosides, neotame, salt of aspartame-acesulfame, polyglycitol syrup, maltitol, maltitol syrup, lactitol, xylitol, erythritol, advantame; (4) emulsifiers, stabilisers, thickeners and gelling agents, such as lecithins, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propane- 1,2-diol alginate, agar, carrageenan, processed eucheuma seaweed, locust bean gum; carob gum, guar gum, tragacanth, acacia gum; gum arabic, xanthan gum, karaya gum, tara gum, gellan gum, konjac, soybean hemicellulose, cassia gum, polyoxyethylene sorbitan monolaurate;Attorney Docket No.:10457-582PC0

[0062] polysorbate 20, polyoxyethylene sorbitan mono-oleate; polysorbate 80, polyoxyethylene sorbitan monopalmitate; polysorbate 40, polyoxyethylene sorbitan monostearate; polysorbate 60, polyoxyethylene sorbitan tristearate; polysorbate 65, pectins, ammonium phosphatides, sucrose acetate isobutyrate, glycerol esters of wood rosins, cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, ethyl methyl cellulose, carboxy methyl cellulose, crosslinked sodium carboxy methyl cellulose, enzymatically hydrolysed carboxy methyl cellulose, sodium, potassium and calcium salts of fatty acids, magnesium salts of fatty acids, mono- and diglycerides of fatty acids, acetic acid esters of mono- and diglycerides of fatty acids, lactic acid esters of mono- and diglycerides of fatty acids, citric acid esters of mono- and diglycerides of fatty acids, tartaric acid esters of mono- and diglycerides of fatty acids, mono- and diacetyltartaric acid esters of mono-and diglycerides of fatty acids, mixed acetic and tartaric acid esters of mono-and diglycerides of fatty acids, sucrose esters of fatty acids, sucroglycerides, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, propane- 1,2-diol esters of fatty acids, thermally oxidised soya bean oil interacted with mono- and diglycerides of fatty acids, sodium stearoyl-2-lactylate, calcium stearoyl-2-lactylate, stearyl tartrate, sorbitan monostearate, sorbitan tristearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, invertase; (5) others, including acid, acidity regulators, anti-caking agents, anti-foaming agents, bulking agents, carriers and carrier solvents, emulsifying salts, firming agents, flavour enhancers, flour treatment agents, foaming agents, glazing agents, humectants, modified starches, packaging gases, propellants, raising agents and sequestrants: acetic acid, potassium acetate, sodium acetate, calcium acetate, lactic acid, carbon dioxide, malic acid, fumaric acid, sodium lactate, potassium lactate, calcium lactate, citric acid, sodium citrates, potassium citrates, calcium citrates, tartaric acid (L-(+)), sodium tartrates, potassium tartrates, sodium potassium tartrate, phosphoric acid, sodium phosphates, potassium phosphates, calcium phosphates, magnesium phosphates, sodium malates, potassium malate, calcium malates, metatartaric acid, calcium tartrate, adipic acid, sodium adipate, potassium adipate, succinic acid, triammonium citrate, calcium disodium ethylene diamine tetra-acetate; calcium disodium EDTA, glycerol, octenyl succinic acid modified gum arabic, diphosphates, triphosphates, polyphosphates, beta-cyclodextrin, stigmasterol-rich plant sterols, sodium carbonates, potassium carbonates, ammonium carbonates, magnesium carbonates, hydrochloric acid, potassium chloride, calcium chloride, magnesium chloride, stannous chloride, sulphuric acid, sodium sulphates, potassium sulphates, calcium sulphate, ammonium sulphate,Attorney Docket No.:10457-582PC0

[0063] aluminium sulphate, aluminium sodium sulphate, aluminium potassium sulphate, aluminium ammonium sulphate, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, magnesium hydroxide, calcium oxide, magnesium oxide, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, sodium aluminium phosphate, silicon dioxide, calcium silicate, magnesium silicate, magnesium trisilicate, talc, sodium aluminium silicate, potassium aluminium silicate, fatty acids, gluconic acid, glucono delta-lactone. sodium gluconate, potassium gluconate, calcium gluconate, ferrous gluconate, ferrous lactate, glutamic acid, monosodium glutamate, monopotassium glutamate, calcium diglutamate, monoammonium glutamate, magnesium diglutamate, guanylic acid, disodium guanylate, dipotassium guanylate, calcium guanylate, Inosinic acid, disodium inosinate, dipotassium inosinate, calcium inosinate, calcium 5 '-ribonucleotides, disodium 5'-ribonucleotides. glycine and its sodium salt, L-leucine, zinc acetate, dimethylpolysiloxane, beeswax, white and yellow, candelilla wax, carnauba wax, shellac, microcrystalline wax, hydrogenated poly- 1 -decene, oxidised polyethylene wax, L-Cysteine. carbamide, argon, helium, nitrogen, nitrous oxide, butane, iso-butane, propane, oxygen, hydrogen, quillaia extract, polydextrose, polyvinylpyrrolidone, polyvinylpolypyrrolidone, polyvinyl alcohol, pullulan, basic methacrylate copolymer, neutral methacrylate copolymer, anionic methacrylate copolymer, polyvinylpyrrolidone-vinyl acetate copolymer, polyvinyl alcohol-polyethylene glycol-graft- co-polymer, oxidised starch, monostarch phosphate, distarch phosphate, phosphated distarch phosphate, acetylated distarch phosphate, acetylated starch, acetylated distarch adipate, hydroxyl propyl starch, hydroxy propyl distarch phosphate, starch sodium octenyl succinate, acetylated oxidised starch, starch aluminium octenyl succinate, triethyl citrate, glyceryl diacetate (diacetin), glyceryl triacetate; triacetin, benzyl alcohol, propan- 1,2-diol; propylene glycol, polyethylene glycol; and (6) colours: curcumin, (i) riboflavin, (ii) riboflavin-5'-phosphate, tartrazine, quinoline yellow, sunset yellow FCF; orange yellow, cochineal; carminic acid; carmines, azorubine; carmoisine, amaranth, ponceau 4R; cochineal Red A, erythrosine, allura red AC, patent blue V, Indigotine; indigo carmine, brilliant blue FCF, chlorophylls and chlorophyllins, copper complexes of chlorophyll and chlorophyllins, green S, plain caramel, caustic sulphite caramel, ammonia caramel, sulphite ammonia caramel, brilliant black BN; black PN, vegetable carbon, brown HT, carotenes, Annatto, bixin, annatto, norbixin, paprika extract; capsanthian; capsorubin, lycopene, beta-apo-8'-carotenal (C30), lutein, canthaxanthin, beetrootAttorney Docket No.:10457-582PC0

[0064] red; betanin, anthocyanins, calcium carbonate, titanium dioxide; iron oxides and hydroxides, aluminium, silver, gold, and litholrubine BK.

[0065] As used herein, the term “phytochemicals” refers to plant-based bioactive compounds produced by plants for their protection. They can be derived from various sources such as whole grains, fruits, vegetables, nuts, and herbs, and more than a thousand phytochemicals have been discovered to date. Non-limiting examples are carotenoids (a-carotene, P-carotene. lutein, lycopene, xanthophylls, cryptoxanthin, fucoxanthin), polyphenols (flavones, flavanones, flavanols, anthocyanidins and anthocyanins, polyphenol amides), isoprenoids (limonene, myrcene, pinene), phytosterols (campesterol, sitosterol, stigmasterol), campestanol, sitostanol, stigmastanol), saponins (dammarane, tirucallane, oleanane), dietary fiber, (pectin, cellulose, lignin, hemicelluloses), and polysaccharides, (amylose, amylopectin, resistant starch, arabinoxylan) (Kumar et al., 2023).

[0066] The term "enhancing muscle functionality" refers to increasing muscle strength, increasing muscle endurance, ameliorating muscle fatigue, improving muscle mass growth and / or reducing muscle recovery time while conducting physical exercise or following physical exercise.

[0067] The term "physical exercise" refers to any physical exertion for a period time by a subject that results in a consumption of energy by skeletal muscle and / or raises the heart rate in a subject that is greater than the consumption of energy by skeletal muscle and / or heart rate in the subject while in a resting state.

[0068] B. Overview

[0069] Generally, obesity refers to abnormal or excessive fat accumulation that presents a risk to health, which is common in people with metabolic syndrome. Obesity makes it difficult for cells to respond to insulin for some reasons not clearly identified yet. Without enough insulin to overcome the resistance, the blood sugar level increases, developing into type 2 diabetes or exacerbating already existing type 2 diabetes. Overall, obesity, metabolic syndrome, and insulin resistance seem to be closely tied, although the causative relationships between them are not clearly established. Obesity is also linked to higher triglyceride levels, higher low-density lipoprotein (LDL, or “bad”) cholesterol levels, and lower high-density lipoprotein (HDL, or “good”) cholesterol levels, which can lead to atherosclerosis, high blood pressure, and stroke.Attorney Docket No.:10457-582PC0

[0070] Obesity and fat accumulation are associated with a higher risk of diabetes and atherosclerotic cardiovascular diseases (Hill and Wyatt, 2013). Lipid accumulation in the muscle induces insulin resistance and impairs glucose uptake (Callahan et al., 2017), while exercise and decreased caloric intake (negative energy balance) are known to decrease fat accumulation and improve glucose and insulin signaling (Heath et al., 1983).

[0071] Generally, “insulin resistance” refers to a diminished sensitivity of target tissues to the metabolic effects of insulin. Insulin resistance reduces glucose uptake and storage, raises blood glucose level, and stimulates compensatory insulin secretion. It is suggested in some studies that obese subjects may have fewer insulin receptors, especially in the skeletal muscle, liver, and adipose tissue, than do lean subjects, however most of insulin resistance appears to be caused by disturbed insulin signaling pathways and consequently abnormal cellular effects such as reduced glucose uptake in insulin-sensitive tissues such as skeletal muscle, liver, and adipose tissues. It is also suggested that insulin signaling disturbance may be closely related to adverse effects of lipid accumulation in tissues such as skeletal muscle and liver as a result of excess weight gain. Due to impaired insulin signaling, obesity may also be related to fat droplets accumulation in skeletal muscle (intramyocellular lipids (IMCLs)). (Schrauwen-Hinderling et al., 2006).

[0072] Repetitive physical exercise induces physiological adaptations in skeletal muscle that improves exercise performance and cognitive function, and pharmacological exercise mimetics inducing such physiologic adaptations may be effective for the treatment of a range of diseases including obesity and metabolic syndrome.

[0073] Genetic evidence indicates that the orphan nuclear receptors estrogen receptor-related receptors (ERRs) play an important role in skeletal muscle exercise capacity. There are three subtypes of ERRs, ERRa, . and y. In a previous study of this lab (Billon et al..2023), it was reported that a synthetic ERR pan agonist, SLU-PP-332, that targets all three ERRs but has the highest potency for ERRa, has sufficient pharmacokinetic properties to be used as an in vivo chemical tool. In the study, the ERR pan agonist increased mitochondrial function and cellular respiration in a skeletal muscle cell line. When administered to mice, the ERR pan agonist increased the type Ila oxidative skeletal muscle fibers and enhanced exercise endurance. It was also observed that the ERR pan agonist induced an ERRa-specific acute aerobic exercise genetic program, and the ERRa activation was critical for enhancing exercise endurance in mice.Attorney Docket No.:10457-582PC0

[0074] In another study of this lab (Billon et al., 2024), the effects of this ERR pan agonist as an exercise mimetic were examined in mouse models of obesity and metabolic syndrome. Diet-induced obese or ob / ob mice were administered with SLU-PP-332, and the effects on a range of metabolic parameters were assessed. SLU-PP-332 administration mimics exercise-induced benefits on whole body metabolism in mice, including increased energy expenditure and fatty acid oxidation. These effects were accompanied by decreased fat mass accumulation. Additionally, the ERR agonist effectively reduced obesity and improved insulin sensitivity in models of metabolic syndrome.

[0075] These results indicate that compounds pharmacologically activating ERR may function as effective exercise mimetics to treat obesity and metabolic syndrome. In this disclosure, another type of ERR activating compounds, i.e., glyceollin I, glyceollin II, and glyceollin III, are introduced. They showed enhanced transcription activator activity of ERRa and ER Ry (Fig. 5). Considering the pharmacological potential of the ERR pan agonist, glyceollins are suggested as exercise mimetics.

[0076] In another study of this lab (Woldt et al., 2013), another family of nuclear receptors were demonstrated to have effects on muscle functionality. REV-ERB a is known to modulate hepatic lipid and glucose metabolism, adipogenesis, and the inflammatory response in macrophages. REV-ERB is a transcriptional repressor, and an REV-ERB agonist increased its transcriptional repression activity. In the study, it was shown that REV-ERB a is highly expressed in oxidative skeletal muscle and plays a role in mitochondrial biogenesis and oxidative function, in gain- and loss-of function studies. Rev-erb-a-deficiency in skeletal muscle leads to reduced mitochondrial content and oxidative function, resulting in compromised exercise capacity. This phenotype was recapitulated in isolated fibers and in muscle cells upon Rev-erb-a knock-down, while Rev-erb-a over-expression increased the number of mitochondria with improved respiratory capacity.

[0077] Regarding the underlying mechanisms, Rev-erb-a-deficiency resulted in deactivation of the Stkll-Ampk-Sirtl-Ppargcl-a signaling pathway, whereas autophagy was up-regulated, resulting in both impaired mitochondrial biogenesis and increased clearance. Over-expression or pharmacological activation of REV-ERB a in muscles with SR9009, the prototype of synthetic agonist, increased respiration and exercise capacity. This study identifies REV-ERB a as a pharmacological target which improves muscle oxidative function by modulating gene networksAttorney Docket No.:10457-582PC0

[0078] controlling mitochondrial number and function. Additional studies have shown that REV-ERB agonists can enhance muscle mass (Mayeuf-Louchart et al., 2017 )

[0079] These results also indicate that compounds such as SR9009 that can pharmacologically activate REV- ERB’s transcription repressor activity may function as effective exercise mimetics to enhance mitochondrial oxidation in skeletal muscle. STL 1267 is a new type of synthetic REVERB agonist. In the study for this disclosure, a mixture of all three types of glyceollins is shown to replace STL 1267 for REV- ERB binding (Fig. 6), suggesting their pharmacological potential as exercise mimetics. Interestingly, individual glyceollins were not as effective as a mixture thereof in functioning as REV-ERB agonists, although they showed REV-ERB transcriptional repressor activity in multiple reporter assays (Fig. 8).

[0080] In summary, the results described above indicate the feasibility of glyceollins (glyceollin I, glyceollin II and glyceollin III) as exercise mimetics that will improve mitochondrial oxidation and muscle function, and reduce obesity. Therefore, in this disclosure, individual and / or combination of glyceollin I, II, and III are suggested to be used as dietary supplements alone or concomitantly with other medicines for the treatment of metabolic syndrome, obesity, insulin resistance, type 2 diabetes, hyperlipidemia of high triglycerides and cholesterol, atherosclerosis, MASH, sarcopenia, osteoporosis, the aging process of muscle and cognition, dementia, and certain cancers.

[0081] C. Glyceollin and AMPK

[0082] Glyceollins, which are soybean phytoalexins, are the isoflavonoids found in soybean seeds that are members of the subfamily Papilionoideae of Fabaceae and known for the high content of isoflavonoids. Isoflavonoids can be divided into several groups based on their structural skeletons; isoflavone, isoflavanone, isoflavanonol, isoflavan, isoflav-3-ene, isoflavan-4-ol, rotenoid and pterocarpan (Fig. 1). Glyceollins are pterocarpans having additional cyclic ether except for glyceollin IV having a prenyl substitution. So far, at least six natural glyceollins have been known. Unlike other isoflavonoids (e.g., daidzein, glycitein, and genistein isoflavones found in soybeans), glyceollins are synthesized only when germinated soybean seeds are infected by fungi or exposed to other pathogens or other types of stress (UV, pH, etc) (Abe et al., 2021).Attorney Docket No.:10457-582PC0

[0083] Some isoflavonoids, e.g., daidzein, and a type of natural phenol, resveratrols (a stilbenoid,), activated ERR in the presence of PGC-ip, which is a transcriptional co-activator of ERR (Uchitomi et al., 2018). Daidzein (DDZ) is a well-known example of nutraceutical supplement, which is isolated from various sources such as soybean, alfalfa, and red clover. It showed a wide range of pharmacological / beneficial properties for diseases such as cardiovascular function in exercise, cholesterol reduction, and anticancer, antifibrotic, and antidiabetic effects. DDZ structure and operation are very similar to those of human estrogens, which are important in preventing osteoporosis, cancer, and postmenopausal diseases (Ubaid et al., 2023).

[0084] Daidzein has also been reported to promote the expression of oxidative phosphorylation-and fatty acid oxidation-related genes via an ERRa- associated pathway to decrease lipid accumulation in muscle cells (Kitamura et al.. 2020). These results suggest that daidzein may be able to enhance the effect of glyceollins on ERRs.

[0085] Although glyceollins have drawn attention due to their anti-estrogenic roles, glyceollins are also known to exert their effect through various signaling pathways independent of estrogen receptors (ERs). AMP-activated protein kinase (AMPK) and insulin signaling pathways are such examples (Wood et al., 2012).

[0086] D. Embodiment examples

[0087] Since ERR transcription activator agonists and REV-ERB transcription repressor agonists enhance skeletal muscle function and mitochondrial fat oxidation even without physical exercise, glyceollins, which are agonists of those nuclear receptors, are expected to have similar effects on skeletal muscle. Thus, the main objective of this invention is to develop compositions comprising glyceollins as food supplements for enhancing skeletal muscle functionality as exercise mimetic.

[0088] In various embodiments, food supplemental products are suggested here, comprising the composition comprising glyceollins alone or in combination with other ingredients, e.g., pharmaceutical excipients, food additives, and conventional dietary supplements such as vitamins, minerals, essential amino acids, protein powder, botanical herbs and compounds.

[0089] As set forth herein, the composition of the invention comprises glyceollin I, glyceollin II, and / or glyceollin III. In some embodiments, the composition comprises glyceollin I about 0.1 mg - 2,000 mg per serving. In some embodiments, the composition comprises glyceollin II about 0.1Attorney Docket No.:10457-582PC0

[0090] mg - 2,000 mg per serving. Tn some embodiments, the composition comprises glyceollin TIT about 0.1 mg - 2,000 mg per serving. Optionally, a total amount of glyceollins (I, IT, and / or ITT) per serving is 1-5,000 mg, or optionally 50-2500 mg. In some embodiments, the composition comprises glyceollin I and glyceollin IT. In some embodiments, the composition comprises glyceollin I and glyceollin TIT. In some embodiments, the composition comprises glyceollin II and glyceollin III. In certain embodiments, the composition comprises all three types of glyceollins, optionally at a ratio of about 1:1:1.

[0091] In some embodiments, the composition may comprise any of phytochemicals selected from daidzein (0.1-25 mg), genistein (0.1-25 mg), glycitein (0.1-25 mg), and resveratrol (0.1-25 mg), and a total quantity of the phytochemicals is less than about 100 mg per serving.

[0092] It would be advantageous to combine glyceollins with daidzein. Daidzein has been reported to activate ERR, which is an estrogen receptor agonist (weak) and is typically not the profile as an exercise mimetic. Since glyceollins, estrogen receptor antagonists, also activate ERRs, glyceollins and daidzein can be combined to eliminate the estrogen receptor effects and enhance the exercise mimetic activity targeting ERR. One key advantage is that daidzein is relatively cheap to manufacture whereas glyceollins are considerably more expensive. Therefore, the cost of the supplements presented here can be reduced.

[0093] Tn some embodiments, the composition may further comprise any of vitamins (including vitamin A, Bi. B2, B3, B5, Be, B7, 69,612, C, D, E. and / or K), minerals (including calcium, chloride, chromium, copper, iodine, iron, magnesium, manganese, molybdenum, phosphorus, potassium, selenium, silicon, vanadium, zinc) and essential amino acids (including histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine).

[0094] Recommended daily intake of vitamins are: vitamin A (700-900 pg), vitamin B 1 (thiamine; 1.1-1.2 mg), vitamin B2 (riboflavin; 1.1-1.3 mg), vitamin B3 (niacin, nicotinic acid; 14-16 mg), vitamin B5 (pantothenic acid; 5 mg ), vitamin B6 (pyridoxal, pyridoxine, pyridoxamine; 1.3 mg-1.7 mg), vitamin B7 (biotin; 30 pg), vitamin B9 (folate, folic acid; 400 pg), vitamin B12 (cobalamin; 2.4 pg), vitamin C (75-90 mg), vitamin D (calciferol; 15-20 pg (600-800 IU)), vitamin E (alpha-tocopherol; 15 mg), vitamin K (phylloquinone, menadione; 90-120 pg), and choline (425-550 mg).Attorney Docket No.:10457-582PC0

[0095] Recommended daily intake of minerals (macro - more than 100 mg per day) for adults are; calcium: 1000-1300 mg / day, magnesium: 310-420 mg / day, sodium: 920-2300 mg / day, potassium: 2800-3800 mg / day, phosphorus: 1000 mg / day, sulfur: 800-900 mg / day, chloride: 1800-2300 mg / day. Essential minerals (trace - less than lOOmg per day) for adults are; chromium: 21-45 mcg / day, iron: 8-27 mg / day, zinc: 8-14 mg / day, copper: 900-1300 mcg / day, manganese: 1800-2600 mcg / day. selenium: 60-75mcg / day, iodine: 150-270 mcg, molybdenum: 45-50 mcg / day, fluoride: 3-4 mg / day, boron: not known; upper tolerable limit: 20 mg / day, silica: 9-14 mg (not known for pregnant and breastfeeding women), and cobalt : 0.006-0.008 mcg / day.

[0096] Recommended daily intake of essential amino acids (EAA) for every 2.2 pounds (about 1 kg) of body weight are: histidine: 14 milligrams, isoleucine: 19 milligrams, leucine: 42 milligrams, lysine: 38 milligrams, methionine: 19mg, phenylalanine: 33 mg, threonine: 20 mg, tryptophan: 5 mg, valine: 24 mg.

[0097] If need be, taurine or live microbials (commonly referred to as “probiotics”) can also be added to the composition.

[0098] In certain embodiments, the composition is formulated into dietary supplement in a solid oral dosage form, and the serving size is 1 mg to 100 g, and optionally less than 10 g.

[0099] Non-limiting examples of excipients for solid oral dosage forms (such as powders, granules, capsules, tablets, pills, dragees (or dragees), modified-release forms of tablets, pills, or capsules (fast / immediate release (IR), slow / sustained release (SR), extended release (ER / XR / XL)), cachets, lozenges (or troches), chewable tablets, orally disintegrating tablets (ODTs), gummies, and films) include: (1) diluents to increase bulk volume of oral dosage forms such as tablet and capsules, such as calcium carbonate, calcium lactate, calcium phosphate, confectioner’s sugar, corn syrups solids, dextrin, dextrose monohydrate / anhydrous, erythritol, isomalt, lactose monohydrate / anhydrous, magnesium carbonate, magnesium hydroxide, magnesium oxide, maltitol, maltodextrin, maltose, mannitol, microcrystalline cellulose (MCC), powdered cellulose, pregelatinized starch (partially), sorbitol, starch, sucrose, trehalose, xylitol, polydextrose, inulin, neusilin, dextrates; (2) binders to form granules or agglomerates and to promote cohesive compacts for directly compressed tablet, such as copovidone, pregelatinized starch (fully), gelatin, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hypromellose (HPMC, hydroxypropyl methylcellulose), maltodextrin, methylcellulose (MC),Attorney Docket No.:10457-582PC0

[0100] polyvinyl alcohol, povidone, sodium alginate, sodium carboxymethyl cellulose (Na CMC), com syrup solids, ethyl cellulose dextrose, starch, sucrose, acacia, tragacanth, agar, colophony, guar gum, isomalt, dextrins, dextrates, zein (maize protein); (3) disintegrants to promote table or capsule breakdown into smaller granules / particles when exposed to aqueous fluids, such as alginic acid, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, low substituted hydroxypropyl cellulose, polacrilin potassium, sodium starch glycolate, starch, pregelatinized starch; (4) controlled release agents for continuously releasing the active ingredients at a predetermined rate over an extended period of time, such as carbomer, carrageenan, chitosan, etylcellulose, fatty alcohols, glyceryl palmitostearate, glyceryl stearate, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, hydrogenated castor oil, hydrogenated vegetable oil, hypromellose acetate succinate, poly(ethylene) oxide, polycarbophil, polyethylene glycol (PEG), polyvinyl acetate and povidone graft copolymer, polyvinyl alcohol, sodium alginate, stearic acid, xanthan gum, methylcellulose, Na carboxymethyl cellulose, modified starch, gellan gum, glyceryl behenate, glyceryl monostearate, povidone, waxes (beeswax, microcrystalline wax, ozokerite wax, paraffin wax); (5) neutral cores (i.e., carriers / substrates for loading of active ingredients for sustained or immediate-release formulations, such as lactose and microcrystalline cellulose (MCC) spheres, MCC spheres, mannitol pellets, sugar spheres, tartaric acid pellets; (6) pH modifiers such as amino acids (L-arginine, L-leucine, glycine, lysine, histidine), glucono delta lactone, lactic acid, potassium chloride, sodium bicarbonate, sodium carbonate, sodium lactate, sodium succinate, calcium carbonate, magnesium carbonate / hydroxide / oxide, potassium hydroxide / citrate / bicarbonate, sodium borate, citric acid monohydrate, ascorbic / tartaric / malic acid, meglumine, orthophosphoric acid, sodium / potassium hydrogen phosphate; (7) encapsulating agent to encapsulate or complex with the actives to make them more water soluble, or to control its release, or stabilize it against oxidative, light, moisture-mediated degradation, such as aliphatic polyesters (polyglycolide. polylactide, and their copolymers), chitosan, cyclodextrin, hydroxypropyl cyclodextrin, ion exchange resins, pea maltodextrin, betadex (beta-cyclodextrin) sulfobutyl ether sodium, alginates, carrageenan, ethylcellulose, cellulose acetate phthalate, shellac; (8) emulsifiers / solubilizers, such as docusate sodium (sodium l,4-bis(2-ethylhexyl) sulfosuccinate or dioctyl sodium sulfosuccinate), glyceryl monostearate / mono- and diglycerides, medium chain triglycerides, poloxamer, polyoxyethylenesorbitan fatty acid esters (polysorbates), polyoxyl 40 hydrogenated castor oil, polyoxylglyceride, sodium lauryl sulfate, sucrose esters, propylene glycolAttorney Docket No.:10457-582PC0

[0101] alginate, cyclodextrins, PEGs; (9) coating agents such as cellulose acetate, cellulose acetate butyrate, cellulose acetate phthalate, ethylcellulose aqueous dispersions, hypromellose phthalate, methyl methacrylate and diethyl / dimethylaminoethyl methacrylate copolymer dispersion, polyvinyl alcohol / PEG graft copolymer, polymethacrylates, polyvinyl acetate dispersion, pregelatinized hydroxypropyl pea starch, Pullulan, shellac, alginate, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, hypromellose acetate succinates, maltitol, methyl cellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, sucrose, ready-to-use coating systems such as Opadry and Sepifilm; (10) plasticizers, on mixing with polymers, to make the form more flexible and elastic, such as acetyl tributyl citrate, acetyl triethyl citrate, dibutyl sebacate, glycerin, propylene glycol, sorbitan ester of fatty acids, sorbitol sorbitan solution, triacetin, tributyl citrate, triethyl citrate, PEGs, polysorbates, sorbitol; (11) colorants such as ferric oxides, titanium dioxide, Brilliant Blue FCF, alizarin cyanine green, quinizarine green SS, dibromo fluorescein, diiodo fluorescein, erythrosine, Ponceau SX, Brilliand Lake Red R, acid fuchsine, Allura red AC and lake. Alizurol Purple SS. tartrazine. Sunset Yellow FCF, fluorescein, naphthol yellow S, Quinoline Yellow WS; (12) lubricants / glidants such as colloidal silicon dioxide and fumed silica, glyceryl behenate, magnesium stearate, Poloxamer 188, sodium stearyl fumarate, sucrose stearate, talc, glyceryl behenate / monostearate, PEGs, hydrogenated castor / vegetable oil, sodium lauryl sulfate, calcium / zinc stearate, stearic acid, sodium benzoate, sodium stearate, leucine; (13) sweeteners such as dextrose, fructose, maltitol, sorbitol, sucrose, trehalose, and xylitol; and acesulfame potassium, aspartame, neohexyl aspartame, sodium saccharin, stevia, sucralose; (14) flavors, taste-masking agent, coprocessed excipients, and polymers for hot-melt extrusion. For capsules, gelatin or hypromellose (hydroxypropyl methylcellulose or HPMC) hard capsules or carrageenan and modified starch soft capsules can be used.

[0102] For example, the composition can be packaged in a vegetable or gelatin capsule, optionally hypromellose (hydroxypropyl methylcellulose or HPMC) capsule, and the capsule may comprise any excipients selected from water, soybean oil, glycerin, beeswax, gelatin, microcrystalline cellulose, hydroxypropyl cellulose, oat fiber, or other plant fiber, rice flour, calcium palmitate, dicalcium phosphate, within a range of 0-50% w / w of a single unit, magnesium silicate and magnesium stearate less than 2% w / w, and other pharmaceutically acceptable excipient which can be a carrier or a diluent.Attorney Docket No.:10457-582PC0

[0103] An example of a capsule for oral administration contains compounds of the invention (for example, 50-2500 mg, or 100-1000 mg), lactose (about 75 mg) and magnesium stearate (about 15 mg). The mixture is passed through a 60 mesh sieve and packed into a gelatin capsule with a size 000 (-1000 mg), 00 (735 mg), 0 (500 mg), 1 (400 mg), 2 (300 mg), 3 (200 mg), or 4 / 5 (less than 200 mg), and optionally 00 size capsule.

[0104] An example of a tablet for oral administration that can be prepared by conventional tableting techniques can contain: for core, active compound (as free compound or salt thereof) (for example, 50-2500 mg, or 100-1000 mg), colloidal silicon dioxide (Aerosil)® (about 1.5 mg), cellulose, microcryst. (Avicel)® (about 70 mg), modified cellulose gum (Ac-Di-Sol)® (about 7.5 mg), and magnesium stearate; and for coating: HPMC approx. 9 mg Mywacett 9-40 T approx. Acylated monoglyceride is used as plasticizer for film coating.

[0105] In certain embodiments, the glyceollin composition is formulated into powders or granules to be packaged into a sachet tea bag or pouch, and the sachet tea bag or pouch optionally comprises powders or granules of botanical herbs or extracts thereof (about 1-5 g), selected from coffee beans, green tea, acai, goji berry, ginkgo, garlic, ginseng, ginger, cinnamon, red date, and / or turmeric (curcuma), some of which are commonly used in alternative medicine in the East Asia, and / or other herbs or botanical compounds. The sachet tea bag containing glyceollin composition with or without botanical herbs or extracts thereof is put into hot water of teacup volume (about 100 -250 m ); or glyceollin composition packaged in a pouch with or without botanical herbs or extracts thereof is poured into hot water of teacup volume (about 100 -250 mL), and the glyceollin composition tea can be taken alone, or with meal or snack, before (optionally within 48 hours), during and / or after (optionally within 48 hours) of physical exercise.

[0106] In certain embodiments, the glyceollin composition is formulated into powders or granules to be mixed with protein powders or granules (about 1-100 g, optionally 20-50 g per serving), which can be optionally formulated into a protein bar (about 1-100 g, optionally 20-50 g per serving). Optionally, the composition may further comprise pharmaceutical excipients for solid oral dosage forms or conventional food additives. The mixture of glyceollin composition and protein powders is suspended in hot water of teacup volume (for example, about 100-250 mL), and the mixture can be taken alone, or with meal or snack, before (optionally within 48 hours), during and / or after (optionally within 48 hours) of physical exercise. Non-limiting examples ofAttorney Docket No.:10457-582PC0

[0107] protein powder that can be mixed with the glyceollin composition are animal protein powders such as casein, whey, collagen, egg white, and insect powders, and plant protein powders extracted from soy. pea, chickpea, hemp, rice, brown rice, white rice, blue-green algae, pumpkin, Sacha Inchi, wheat, buckwheat, potato protein, and fiber and protein rich amaranth sprout.

[0108] In certain embodiments, the composition is formulated into a liquid oral dosage form, in about 1-100 mL, optionally 10-50 mL. Also, the composition may further comprise vitamins, minerals, essential amino acids, phytochemicals, botanical herbs or extracts thereof, taurine, caffeine, carbohydrates, other pharmaceutical excipients for liquid oral dosage forms, and / or conventional food additives.

[0109] Non-limiting examples of excipients for liquid oral dosage forms (such as solutions, syrups, suspensions, elixirs, emulsions, drops) include: (1) vehicles to dilute actives to a suitable volume, such as confectioner’s sugar, glucose syrup, maltitol solution, com syrup solids, dextrose, glycerin, lactose, maltitol, maltodextrin, mannitol, propylene glycol, sorbitol, sucrose, trehalose, xylitol; (2) suspending agents and thickeners such as magnesium aluminum silicate, microcrystalline cellulose and Na carboxymethyl cellulose, propylene glycol alginate, acacia, carbomer, carrageenan, colloidal silicon dioxide, gellan gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, maltitol, methyl cellulose, pectin, PEGs, polyvinyl alcohol, povidone, Na carboxymethyl cellulose, sorbitol, sucrose, xanthan gum, tragacanth, gelatin, guar gum, kaolin, phospholipids; (3) emulsifiers / solubilizers such as 2-pyrrolidone, lecithin / phospholipids, polyoxyl glycerides, propylene glycol; (4) antioxidants such as ascorbic acid, butylated hydroxyl anisole, butylated hydroxyl toluene, sodium ascorbate, sodium metabisulfite, sodium sulfite, tocopherol; (5) preservatives such as methyl paraben, propyl paraben, butyl paraben, potassium sorbate, EDTA, cetrimide, benzyl alcohol, thimerosal, phenylmercuric salts, benzalkonium chloride; (6) pH modifiers such as amino acids (L-arginine, L-leucine, glycine, lysine, histidine), glucono delta lactone, lactic acid, potassium chloride, sodium bicarbonate, sodium carbonate, sodium lactate, sodium succinate, calcium carbonate, magnesium carbonate / hydroxide / oxide, potassium hydroxide / citrate / bicarbonate, sodium borate, citric acid monohydrate, ascorbic / tartaric / malic acid, meglumine, orthophosphoric acid, sodium / potassium hydrogen phosphate; (7) encapsulating agents such as aliphatic polyesters (polyglycolide, polylactide, and their copolymers), chitosan, cyclodextrin, hydroxypropyl cyclodextrin, ionAttorney Docket No.:10457-582PC0

[0110] exchange resins, pea maltodextrin, betadex (beta-cyclodextrin) sulfobutyl ether sodium, alginates, carrageenan, ethylcellulose, cellulose acetate phthalate, shellac; and (8) flavors and taste masking agent.

[0111] In some embodiments, glyceollin-protein shake blend or glyceollin liquid may further comprise any of the following ingredients for weight-loss; African mango, beta-glucans, bitter orange, caffeine, calcium, capsaicin, carnitine, chitosan, chromium, coleus forskohlii, conjugated linoleic acid, fucoxanthin, garcinia cambogia, glucomannan, green coffee bean extract, green tea and green tea extract, guar gum, guarana, hoodia, kola (or cola) nut, mate, probiotics, pyruvate, raspberry ketone, vitamin D, white kidney bean / bean pod, yerba mate, yohimbe (https: / / ods.od.nih.gov / factsheetsAVeightLoss-Consumer / ).

[0112] In some embodiments, glyceollin-protein shake blend or glyceollin liquid may further comprise any of the following ingredients for exercise and athletic performance; antioxidants (vitamin C, vitamin E, and coenzyme Q 10), arginine, beetroot or beet juice, beta-alanine, beta-hydroxy-beta-methylbutyrate (HMB), betaine, branched-chain amino acids (BCAAs). caffeine, citrulline, creatine, deer antler velvet, dehydroepiandrosterone (DHEA), ginseng, glutamine, iron, protein, quercetin, ribose. sodium bicarbonate, tart or sour cherry, Tribulus terrestris (https: / / ods.od.nih.gov / factsheets / ExerciseAndAthleticPerfomiance-Consumer / ).

[0113] In another embodiment, the glyceollin composition is formulated into powders or granules to be reconstituted for injection or into an injection solution (optionally about 0.1 -5 mL volume), and the composition may further comprise pharmaceutical excipients for parenteral dosage forms.

[0114] Non-limiting examples of excipients for parenteral dosage forms (solution, suspension, emulsion, lyophilized or sterile powders for reconstitution suitable for administration by injection, infusion, or implantation into the human body) include: (1) lyoprotectants such as (high purity and low endotoxin quality) human albumin, lactose monohydrate, maltose / trehalose / sucrose (low endotoxin), mannitol (low endotoxin), dextran, inulin, fructose; (2) encapsulating agents such as aliphatic polyesters (polyglycolide, polylactide, and their copolymers), phospholipids / lecithin (phosphatidic acids, phosphoglycerols, phosphoserines, phosphoethanolamines, phosphocholines, PEGylated phospholipids, hydroxypropyl cyclodextrin, betadex (beta-cyclodextrin) sulfobutyl ether sodium; (3) solubilizers and emulsifiers such as N-methyl 2-pyrrolidone, PEG, polysorbate, polyoxyl 35 castor oil, polyoxyl-15-hydroxystearate, polyvinyl pyrrolidone, propylene glycol,Attorney Docket No.:10457-582PC0

[0115] sodium cholesteryl sulfate, sorbitan esters, poloxamers, 2-pyrrolidone, di acyl glycerols, monoglycerols; (4) tonicity agents such as dextrose, glycerin, mannitol, KCl / NaCl, sorbitol; (5) solvents and cosolvents such as water immiscible (including ethyl oleate, benzyl benzoate, vegetable oil, soybean oil, sesame oil, peanut oil, castor oil, almond oil, cottonseed oil) and water miscible (including propylene glycol, PEG low molecular weight, glycerin, ethanol. 2-pyrrolidone, N-methyl-2-pyrrolidone); (6) viscosity-building agents such as sodium carboxymethyl cellulose (Na-CMC), methylcellulose (MC), gelatin, polyvinyl pyrrolidone; (7) antioxidants such as ascorbic acid, acetylcysteine, sodium ascorbate, sodium metabisulfite, sodium bisulfite, tocopherol; (8) chelating agent EDTA; (9) preservatives such as methyl paraben, propyl paraben, benzalkonium chloride, thimerosal, phenyl mercuric salts; and (10) buffering agents such as acetate, citrate, tartarate, phosphate, triethanolamine (TRIS) buffer. A unit dosage form for injection can be in ampoules or in multi-dose containers.

[0116] An example of injectable preparation is produced by aseptically placing, for example, 50-2500 mg, or 100-1000 mg of the glyceollin composition into a vial, aseptically freeze-drying and sealing. For use, the contents of the vial are mixed with 0.1 - 5 mL of sterile physiological saline or the like, to produce an injectable preparation.

[0117] In another embodiment, the composition is formulated for topical administration, including transdermal patch, gels, creams, ointments, or spray, and the composition further comprises necessary pharmaceutical excipients.

[0118] Non-limiting examples of excipients for topical dosage forms (such as ointments, creams, gels, pastes, lotions, jellies, and poultices) include: (1) gelling agents such as carbomer, carrageenan, chitosan, gelatin, gellan gum, pectin, poloxamer, poly(ethylene) oxide, polycarbophil, pullulan, hydroxyethyl cellulose, hypromellose, methyl cellulose, alginates, Na carboxymethyl cellulose, xanthan gum, acacia, agar, guar gum, tragacanth, modified starch, povidone; (2) humectants such as corn syrup solids, glycerin, lactic acid, PEGs, propylene glycol, sodium lactate, sorbitol, trehalose, xylitol; (3) cream and ointment bases such as cetostearyl alcohol (type A), cetyl palmitate, fatty alcohols, hard fat, lanolin, lanolin alcohol, hydrogenated castor oil, mineral oil, petrolatum, glyceryl behenate, hard paraffin, soft paraffin, stearic acid, beeswax (white and yellow), carnauba wax, emulsifying wax, microcrystalline wax; (4) emulsifiers / solubilizers such as docusate sodium (sodium l,4-bis(2-ethylhexyl) sulfosuccinate or

[0119] 1Attorney Docket No.:10457-582PC0

[0120] dioctyl sodium sulfosuccinate), glyceryl monostearate / mono- and diglycerides, medium chain triglycerides, poloxamer, polyoxyethylenesorbitan fatty acid esters (polysorbates), polyoxyl 40 hydrogenated castor oil, polyoxylglyceride, sodium lauryl sulfate, sucrose esters, propylene glycol alginate, cyclodextrins, PEGs, 2-pyrrolidone, lecithin / phospholipids, polyoxyl glycerides, propylene glycol, cocoyl caprylocaprate, decyl oleate, diethylene glycol monoethyl ether, dimethyl isosorbide, glyceryl monooleate, isopropyl myristate. MCT (Medium Chain Triglyceride), octyldodecanol, oleyl alcohol, polyoxyethylene alkyl ethers, polyoxyethylene stearates, propylene glycol monocaprylate, propylene glycol monolaurate, sodium cetostearyl sufate, lecithin, cyclodextrins, docusate sodium, glyceryl monostearate, hydrogenated vegetable / cottonseed / palm kernel oil, MCT, N-methyl-2-pyrrolidone, poloxamer, polysorbate, PEG, PEG castor oil derivatives, propylene glycol, polyoxyl glycerides, sodium lauryl sulfate, sucrose esters; (5) suspending agents / thickeners such as magnesium aluminum silicate, microcrystalline cellulose and Na carboxymethyl cellulose, propylene glycol alginate, acacia, carbomer, carrageenan, colloidal silicon dioxide, gellan gum. hydroxyethyl cellulose, hydroxypropyl cellulose, hypromellose, maltitol, methyl cellulose, pectin, PEGs, polyvinyl alcohol, povidone, Na carboxymethyl cellulose, sorbitol, sucrose, xanthan gum, tragacanth, gelatin, guar gum, kaolin, phospholipids; (6) preservatives such as methyl paraben, propyl paraben, butyl paraben, potassium sorbate, EDTA, cetrimide, benzyl alcohol, thimerosal, phenylmercuric salts, benzalkonium chloride, ethyl paraben, benzoic acid, sodium benzoate, potassium sorbate, imidurea; (7) pH modifiers such as lactic acid, citric acid, tartaric acid, ascorbic acid, and their sodium salt; (8) antioxidants such as ascorbic acid, butylated hydroxyl anisole, butylated hydroxyl toluene, sodium ascorbate, sodium metabisulfite, sodium sulfite, tocopherol; and (9) chelating agent EDTA.

[0121] In another embodiment, the composition is formulated into compressed pellets to be placed in the subcutaneous area of the lower abdomen or buttocks, and the composition may further comprise pharmaceutical excipients for solid dosage forms. For application of compressed pellets to be implanted subcutaneously as depot injections, such implants can employ known inert materials such as silicones and biodegradable polymers, e.g., polylactide- polyglycolide. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides).Attorney Docket No.:10457-582PC0

[0122] More detailed information on the excipients and conventional techniques for preparation are described in Remington: The Science and Practice of Pharmacy, 23rd Ed., 2021, or later versions thereof.

[0123] Also, a method of use of the composition comprising glyceollins is suggested for enhancing muscle functionality in a subject. The method comprises a step of administering an effective amount of the aforementioned composition, optionally comprising all three glyceolins at a ratio of about 1:1:1, preferably through oral route, although subcutaneous or intravenous injection may be contemplated. The composition can be administered alone or with meal or snack, (e.g., 48 hrs) before, during, and / or (e.g., 48 hrs) after of physical exercise. A daily dose of a total amount of glyceollins (I, II, and / or III) is less than 5000 mg; and the composition comprising glyceollins can be administered daily, or more than once a day, such as twice or thrice daily. Alternatively, the composition can be administered less frequently than daily, such as every other day, twice a week, or once a week, if found to be advisable by a prescribing physician.

[0124] Further, the glyceollin composition can be co-administered as adjuvant treatment along with other surgical or pharmaceutical intervention for the treatment of metabolic syndrome, obesity, insulin resistance, type 2 diabetes, hyperlipidemia of high triglycerides and high cholesterol, atherosclerosis, metabolic associated steatohepatitis (MASH), sarcopenia, osteoporosis, the aging process of muscle and cognition, dementia, and certain cancers.

[0125] On one aspect, it can also be contemplated to include other types of glyceollins (e.g., glyceollin Vi, glyceollin, V, and glyceollin VI) in the inventive composition.

[0126] EXAMPLES

[0127] Example 1. Materials and Methods

[0128] 1.1. Glyceollin preparation

[0129] There are two options to produce proprietary soy compound(s) as dietary supplements. Glyceollins can be chemically synthesized through 7 to 10 steps as described in Luniwal et al. (2011). However, chemical synthesis is not environmentally friendly, so they can be purified from the soybeans as described in Abe et al. (2021).

[0130] 1.2. Promoterreporter cloningAttorney Docket No.:10457-582PC0

[0131] Bmall::luc was constructed as follows: Sequences (Bmal (nt 1-1940), CMV (nt 941-1682), Iuc2-P2A-Nluc (1786-4140) were constructed into the plasmid backbone (Promega, ref:N1481 pNLCoI3[luc2-P2A-NlucP / CMV / Hygro).

[0132] 3XERRE PGK::nluc was constructed as follows: Sequences (ERRE (tcaaggtca) 3XERRE (nt 4415-4173), PGK (nt 4181-4695) , Iuc2-P2A-Nluc (4731-7084) were constructed into the plasmid backbone (Promega, RefN1491 pNLCoI4[luc2-P2A-NlucP / PGK / Hygro]). The 0XERRE PGK::nluc construct is the base construct without the ERRE insert.

[0133] CYP8Bl::nluc was constructed as follows: Sequences (CYPB1 (nt 3493-5048), miniP (nt 5094-5124), Iuc2-P2A-Nluc (5157-7511) were constructed into the plasmid back bone Promega, ref:N1471 pNLCoI2[luc2-P2A-NlucP / minP / Hygro]).

[0134] SLC10AE.:nluc was constructed as follows: Sequences (SlclOal (nt 3493-4895), miniP (nt 4941-4971), Iuc2-P2A-Nluc (5004-7358) were constructed into the plasmid backbone Promega, ref:N1471 pNLCoI2[luc2-P2A-NlucP / minP / Hygro]).

[0135] 1.3. Cotransfection Assays:

[0136] For REV-ERB activity assay, either REV-ERBa or REV-ERB£ full length expression vectors were transfected into HEK293 cells along with Bmall::luc, CYP8bl::luc, or SLC10Al::luc reporter plasmids using Lipofectamine 2000 and standard protocols provided by the lipofectamine manufacturer (Promega). GFP plasmid was also in the transfection mix to confirm transfection efficiency. HEK cells were plated in 10 cm dishes for western blot analysis, and HEK UAS-luc cells were plated in 96 well plates for luciferase assay analysis. Prior to cellular adherence, transfection mix was added. The cells and transfection mix were incubated at 37°C in a humidified atmosphere of 5% CO2. For luciferase assays, glyceollins were added in an 11-point serial dilution the day following overnight transfection. Cells were incubated with the compound for 24 h, prior to luciferin addition and luminescence read out.

[0137] For ERR activity assay, HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum at 37°C under 5% CO2. Twenty-four hours prior to transfection, HEK293 cells were plated in 96-well plates at a density of 2 x 104cells / well. ERRa or ERRy full-length proteins were overexpressed with expression vectors using cotransfection protocols as described for the REV-ERBs above. 0XERRE PGK::luc and 3XERREAttorney Docket No.:10457-582PC0

[0138] PGK::luc reporters were used, individually, in the cotransfection assays to assess specific ERR activity vs, any potential non-ERR specific activity.

[0139] 1.4. Radioligand Displacement Binding Assay

[0140] The DNA sequences expressing human REV-ERBa LBD (a.a. 281-614) and human REV-ERBb LBD (a.a.381-579) were cloned into a pRLP121 plasmid (ampR) using NcoI / BamHI and Ncol / Xhol restriction sites, respectively. E.coli BL21(DE3) cells (Thermo-Fisher) carrying the expression vector were grown in LB containing Ampicillin (lOOpg / mL). Expression was induced for 20 h at 16°C by addition of 1 mM IPTG (Millipore-Sigma) once the OD reached 0.6-0.8. The media was supplemented with 150 mM 5 -Aminolevulinic Acid Hydrochloride (Millipore-Sigma) during the induction process. 5-ALA is a heme precursor and is needed for REV-ERB protein stability. Cells were harvested by centrifugation at 6,000 g for 15 minutes at 4°C. Cells from 1 liter of culture were resuspended in 30mL of lysis buffer (25 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 5 mM DTT, 10 pg / mL lysozyme (Roche), 5 ug / mL DNase grade I (Roche)) and stored at -80°C until purification. Cell disruption was performed by sonication (Branson, SFX550), at 4°C, with three rounds of 2 minutes each (0.5s on - 0.5s off) at 30% intensity with 2 minutes of incubation on ice in between rounds. After disruption, cells were centrifuged for 20 minutes at 20,000 g after which proteins were present in the supernatant (soluble fraction). The purification process was performed using an AKTA pure 25 M (GE Healthcare) FPLC system. Buffer A (25 mM Tris pH 8.0, 500 mM NaCl, 10% glycerol, 1 mM DTT) and buffer B (buffer A containing 500 mM imidazole) were used during this process. The soluble fraction was injected on an IMAC column (5 mL HisTrap HP, GE Healthcare) equilibrated with 4% buffer B. The column was washed with 4% buffer B (8 CV) and then washed with 10% buffer B (8 CV). Elution was executed by a linear gradient from 10% to 100% of buffer B (12 CV) with 4 ml fractions collected. Protein purity was analyzed by SDS-PAGE (MiniProtean TGX 4-20% 15 well gels, Bio-Rad) and colored using Coomassie blue. The purest fractions from the affinity chromatography were injected on SEC (HiLoad 16 / 600 Superdex 200, GE Healthcare) equilibrated with buffer A. Fractions were set to be collected every 1 ml and the purity was analyzed by SDS-PAGE as described above. The purest fractions were pooled, then concentrated if needed, and aliquoted to be stored at -80°C for later use in biochemical assay. Amicon Ultra filtration tubes (Millipore-Sigma) were used for protein concentration steps. Scintillation proximity assays (SPA) were runAttorney Docket No.:10457-582PC0

[0141] in 96-well NBS microplates (Coming) with a final volume of 120 pl per well. The tritiated ligand3H-STL1267 was produced by Moravek Biochemicals (Brea, CA, USA). Copper His-TAG YSI SPA Beads (Perkin Elmer) was used. The conditions for the displacement assay are based on the saturation assay results. For this assay3H-STL1267 and REV-ERB LBDs concentrations remained constant and performed a dose response of potential unlabeled competitors for3H-STL1267. The concentration of3H-STL1267 and His-tagged LBD were 80 nm and 120 nM, respectively, for REV-ERBa and 30 nM and 60 nM for REV-ERBb. The same quantity of SPA beads was used for the saturation and for the displacement assay. The dose response concentration started at 100 pM for 11 points with 1 / 3 serial dilution. Thirty 30 pl of test compounds diluted in assay buffer (4X final concentration) were added to the plate first, followed by 30 pl of Elis-Tagged LBD / 3H-STL1267 mixture (4X final concentration). After 30 minutes of incubation at room temperature, 240 ng of SPA beads resuspended in assay buffer was added to each well. Plates were read in a Microbeta2 Plate Reader (Perkin Elmer) after a 20-minute incubation at room temperature on a rocker protected from the light and an extra 10 minutes without agitation. Results were plotted in GraphPad Prism using the signal in CPM with “Binding-Competitive: One site - Fit Ki”.

[0142] 1.5. Animal model

[0143] Male C57B16 mice (n=15-16 / group; 8-10 weeks of age) were subcutaneously administered with glyceollin (20 mg / kg) or vehicle (10% DMSO, 5% Cremophor EL, 5% Tween80, 80% PBS) once per day for 30 days. On day 30 mice were subjected to a maximal aerobic exercise test performed on a treadmill (Columbus) after adequate acclimation via short walking sessions at a low speed of 2m / min for 10 min / day. The maximal aerobic exercise capacity test began with 2 warm-up steps of 2 min at 10 m / min and 6 min at 12 m / min for 6 minutes immediately followed by a classic progressive maximal exercise test protocol increasing the speed by 2 m / min every 2 min until exhaustion. Exhaustion was determined by a lack of responsiveness to 10 consecutive 3-ms electrical shocks. The time from the initiation of the warm-up to exhaustion was recorded. Differences between the two groups were assessed by a Student’s t test (2-tailed). **, p<0.01.

[0144] Example 2. Glyceollins Activate ERR Transcriptional Activator Activity.

[0145] With reference to FIG. 5, shown is (a) Schematic of the ERR cell-based cotransfection assay in HEK293 cells. Cells were transfected with an expression vector for ERR (either humanAttorney Docket No.:10457-582PC0

[0146] ERRa and human ER Ry) along with a luciferase reporter plasmid containing a promoter with 3 copies of a ERRE DNA response element that confers ERR responsiveness to the luciferase reporter (nanoluciferase - nine). As a control to confirm ERR dependency of modulation of the luciferase reporter, HEK293 cells were transfected with a reporter identical to the reporter described above but lacking the ERRE DNA response elements, (b) Glyceollin I, II and III display dose-dependent ERRa and ERRy agonist activity. Cells cotransfected as described in (a) were treated with various doses of glyceollins and an increase in reporter gene activity was observed. Activity in the ERRE-reporter assay was normalized to any activity observed with the reporter lacking the ERRE reporter to determine the level of activity generated by targeting the ERRs. (c) Example of activity of known ERR activators, SLU-PP-332 and SLU-PP-915, in the ERRa and ERRy cotransfection assays. Comparison of the level of activity of ERR agonist exercise mimetics SLU-PP-332 and SLU-PP-915 to the glyceollins indicates they have similar level of ability to activate the receptors and drive ERR-dependent target gene transcription. A / B=amino terminal domain, DBD=DNA binding domain, LBD=Ligand binding domain.

[0147] Example 3. Glyceollins are REV-ERB Ligands.

[0148] With reference to FIG. 6, glyceollins I, II, III and a mixture of I, II and III were used assessed for their ability to displace a radiolabeled REV-ERB agonists,3H-STL1267. STL1267 is a high affinity REV-ERB ligand and the ligand displacement assay displayed in this figure can be used to determine if an added chemical compound can bind to the REV-ERB receptor and displace the high affinity radiolabeled REV-ERB ligand. Radioligand displacement assays for both REV-ERBa (a) and REV-ERBP (b) were performed with the individual glyceollins or the mixture. While individual glyceollins were ineffective in displacing the radioligand and behaving as REVERB ligands, the mixture of glyceollins (I, II and III) were very effective in displacing the radioligand and thus behaved as a REV-ERB ligand.

[0149] Example 4. Glyceollins Activate REV-ERB Transcriptional Repressor Activity.

[0150] With reference to FIG. 7, REV-ERBs are ligand-dependent transcriptional repressors and thus activators or agonists of these receptors drive repression of target gene transcription. Based on the activity of glyceollin in the radioligand binding assay shown in Fig. 6, their functional activity in a cell-based cotransfection reporter assay was assessed, (a) Schematic illustrating theAttorney Docket No.:10457-582PC0

[0151] three luciferase plasmid constructs used in the REV- ERB cotransfection assay. HEK293 cells were contransfected with an expression vector driving the expression of human REV-ERB (REVERB a) along with 1 of three luciferase reporter vectors. Each of these reporter vectors were designed to be driven by natural promoters that are known to be REV-ERB regulated via REVERB DNA response elements and include the promoters derived from the following genes. Bmall, SlclOal, and Cyp8bl. (b) Glyceollins function as REV-ERB agonists in the REV-ERB / Bmall-luciferase reporter cotransfection assay. The glyceollins dose-dependently suppress REV-ERB driven Bmall reporter gene transcription thus functioning as REV-ERB agonists. The mixture of glyceollins function more potently than any of the individual glyceollins. (c) Glyceollins function as REV-ERB agonists in the REV-ERB / Cyp8bl promoter-luciferase reporter cotransfection assay. The glyceollins dose-dependently suppress REV-ERB driven Cyp8bl reporter gene transcription thus functioning as REV-ERB agonists. The mixture of glyceollins function more potently than any of the individual glyceollins. (d) Glyceollins function as REV-ERB agonists in the REV-ERB / SlclOal promoter-luciferase reporter cotransfection assay. The glyceollins dose-dependently suppress REV-ERB driven SlclOal reporter gene transcription thus functioning as REV- ERB agonists. The mixture of glyceollins function more potently than any of the individual glyceollins. A / B=amino terminal domain, DBD=DNA binding domain, LBD=Ligand binding domain.

[0152]

[0153] of Glyceollin to the Exercise Mimetic SR9009 in Biological Assays With reference to FIG. 8, SR9009 is a prototypical REV-ERB agonist and has demonstrated exercise mimetic activity in vivo (Nature Medicine 2013). The activity of glyceollins (mixture of I, II and III) were compared with SR9009 in the Bmall promoter - luciferase REV- ERB a cotransfection assay, and a comparison of the activity of these two types of compounds is shown in the figure. Although SR9009 is somewhat more potent, glyceollin is considerably more efficacious. These results are consistent with the activity of glyceollin consistent with a known exercise mimetic that targets REV- ERB.

[0154] 6. Glyceollin administration increases exercise endurance in mice.

[0155] Male C57B16 mice 8-10 weeks old (N=15-16 / group) were administered vehicle (PBS / Tween 80 / Cremophor EL / DMSO (80% / 5% / 5% / 10%)) or 20 mg / kg glyceollin once per day for 30 days subcutaneously. Glyceollin administration results in a 22% increase in exerciseAttorney Docket No.:10457-582PC0

[0156] endurance in mice (FIG. 9). This data provides clear utility of glyceollin as an exercise mimetic, which is consistent with these compounds functioning as REV-ERB / ERR agonists.

[0157] Example 7. Glyceollin administration effects on mitochondria

[0158] Figure 10 provides a graph showing the effects of glyceollin administration on mitochondrial respiratory gene expression. It was shown that administration of glyceollin produces a coordinated enhancement of mitochondrial respiratory gene expression and aerobic exercise capacity, establishing glyceollin as a previously unrecognized modulator of mitochondrial function and energy metabolism. In vivo administration of glyceollin to mice resulted in a substantial increase in exercise endurance (-22% improvement in maximal treadmill performance), indicating enhanced oxidative capacity. Consistent with this physiological effect, molecular analysis revealed coordinated induction of genes encoding core subunits of mitochondrial complex I, including the mitochondrially encoded genes MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND5, and MT-ND6, as well as the nuclear-encoded complex I subunit NDUFV1. The simultaneous upregulation of both mitochondrial and nuclear respiratory chain genes indicates that glyceollin promotes a systemic enhancement of mitochondrial respiratory function rather than isolated transcriptional changes in the mitochondrial genome. This coordinated transcriptional activation suggests that glyceollin increases mitochondrial oxidative capacity and supports enhanced ATP generation during sustained energy demand.

[0159] These findings establish glyceollin and related compounds as exercise mimetics capable of improving mitochondrial function and metabolic performance independent of physical training. The ability of glyceollin to increase expression of multiple mitochondrial respiratory chain components and to enhance endurance performance demonstrates a previously unrecognized utility of glyceollin in promoting mitochondrial health, mitochondrial biogenesis or functional capacity, and oxidative metabolism. Accordingly, glyceollin may be used to improve mitochondrial function and energy metabolism in a variety of contexts, including enhancement of physical performance, preservation of muscle function, treatment or prevention of disorders associated with impaired mitochondrial function, and improvement of metabolic health. The discovery that glyceollin simultaneously enhances mitochondrial respiratory gene expression and exercise endurance provides a novel and unexpected basis for therapeutic, nutraceutical, andAttorney Docket No.:10457-582PC0

[0160] performance-enhancing applications targeting mitochondrial function and systemic metabolic fitness.Attorney Docket No.:10457-582PC0

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Claims

Attorney Docket No.:10457-582PC0CLAIMSWhat is claimed is:What is claimed is:

1. A composition for enhancing muscle functionality and / or mitochondrial function, comprising at least two glyceollins, selected from:a. glyceollin I,b. glyceollin II, andc. glyceollin III,wherein the quantity of glyceollin I in the composition is within a range of 0.1-2,000 mg per serving;wherein the quantity of glyceollin II in the composition is within a range of 0.1-2,000 mg per serving;wherein the quantity of glyceollin III in the composition is within a range of 0.1-2,000 mg per servingwherein optionally the composition comprises all three glyceollins at a ratio of about 1:1:1; and wherein the composition comprises a total quantity of glyceollins at a range of 1- 5000 per serving, optionally 50 - 2500 per serving; andwherein the serving comprises 10-10,000 mg.

2. The composition of claim 1, wherein the composition optionally further comprises at least one phytochemical selected from daidzein (0.1-25 mg), genistein (0.1-25 mg), glycitein (0.1-25 mg), and resveratrol (0.1-25 mg), and wherein a total quantity of the phytochemical(s) is less than about 100 mg per serving.

3. The composition of claim 1 or 2, wherein the composition optionally further comprises any of vitamins, minerals, and / or essential amino acids.

4. The composition of claim 1, 2 or 3, wherein the composition is formulated into dietary supplement in a solid oral dosage form such as powders, granules, capsules, tablets, pills, dragees (or dragees), modified-release forms of tablets, pills, or capsules (fast / immediate release (IR), slow / sustained release (SR), extended release (ER / XR / XL)), cachets, lozenges (or troches), chewable tablets, orally disintegrating tablets (ODTs), gummies, and films, and wherein the serving size of the dosage form is 1 mg to 100 g.Attorney Docket No.:10457-582PC05. The composition of claim 4, wherein the composition optionally comprises necessary excipients suitable for the solid oral dosage form.

6. The composition of claim 4 or 5, wherein the composition is packaged in a vegetable or gelatin capsule, optionally hypromellose (hydroxypropyl methylcellulose) capsule, comprising any of water, soybean oil, glycerin, beeswax, gelatin, microcrystalline cellulose, hydroxypropyl cellulose, oat fiber, or other plant fiber, rice flour, calcium palmitate, di-calcium phosphate, within a range of 0.1-50 % w / w of a serving size, and / or magnesium silicate and magnesium stearate less than 2 % w / w of a serving size.

7. The composition of claim 1, 2 or 3, wherein the composition is formulated into powders or granules to be packaged into a sachet tea bag or pouch.

8. The composition of claim 7, wherein the sachet tea bag or pouch optionally comprises powders or granules of botanical herbs or extracts thereof, selected from coffee beans, green tea, acai, goji berry, ginkgo, garlic, ginseng, ginger, cinnamon, red date, and / or turmeric (curcuma).

9. The composition of claim 7 or 8, wherein a sachet tea bag containing glyceollin composition with or without botanical herbs or extracts thereof is put into hot water of teacup volume (about 100 -250 mL); or glyceollin composition packaged in a pouch with or without botanical herbs or extracts thereof is poured into hot water of teacup volume (about 100 -250 mL).

10. The composition of claim 1, 2, or 3, wherein the composition is formulated into powders or granules to be mixed with protein powders or granules (about 1-100 g), which can be optionally formulated into a protein bar (about 1-100 g), wherein the composition optionally further comprises pharmaceutical excipients for solid oral dosage forms or conventional food additives.

11. The composition of claim 1, 2 or 3, wherein the composition is formulated into a liquid oral dosage form, optionally in 1-100 mL, wherein the composition optionally further comprises vitamins, minerals, essential amino acids, phytochemicals, botanical herbs or extracts thereof, taurine, caffeine, carbohydrates, and / or other pharmaceutical excipients for liquid oral dosage forms or conventional food additives.

12. The composition of claim 1, 2 or 3, wherein the composition is formulated into powders or granules to be reconstituted for injection or into an injection solution for parenteral administration, and wherein the composition further comprises pharmaceutical excipients for parenteral dosage forms.

13. The composition of claim 1, 2 or 3, wherein the composition is formulated for topical administration, including transdermal patch, gel, cream, or spray, and the composition further comprises necessary pharmaceutical excipients.Attorney Docket No.:10457-582PC014. The composition of claim 1 , 2 or 3, wherein the composition is formulated into compressed pellets to be placed in the subcutaneous area of the lower abdomen or buttocks, and wherein the composition further comprises pharmaceutical excipients for solid dosage forms.

15. A method of use of the composition comprising glyceollins of any of claims 1 to 11 for enhancing muscle functionality and / or mitochondrial function in a subject, the method comprising a step of administering an effective amount of a composition through oral administration.

16. The method of claim 15, the method comprising administering the composition alone or with meal or snack, before, during, and / or after of physical exercise.

17. The method of claim 15 or 16, wherein a daily dose of glyceollins in the composition is less than 5000 mg.

18. The method of any of claims 15 to 17, wherein the composition is administered once, twice, thrice a day, every other day, twice a week, or once a week.

19. A method of use of the composition comprising glyceollins of any of claims 1 to 13, wherein the glyceollin composition is co-administered with a surgical or pharmaceutical intervention for the treatment of metabolic syndrome, obesity, insulin resistance, type 2 diabetes, hyperlipidemia of high triglycerides and high cholesterol, atherosclerosis, metabolic associated steatohepatitis (MASH), sarcopenia, osteoporosis, the aging process of muscle and cognition, dementia, and certain cancers.

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