Compositions and methods that improves metabolic health, gut health, longevity, and stress resilience

Abstract

Provided herein are methods and compositions for improving the health of a subject including metabolic health, gut health, longevity, and stress resilience.

Description

Docket No. NEM-103-PCT Customer No. 51079 TTTLE COMPOSITIONS AND METHODS THAT IMPROVES METABOLIC HEALTH,GUT HEALTH, LONGEVITY, AND STRESS RESILIENCE BACKGROUND

[0001] The invention generally relates to a composition and method of improving metabolic health, improving gut health, improving longevity, and stress resilience.

[0002] Metabolic health and systemic stress resilience are fundamental determinants of human health, functional longevity, and risk of disease. Impaired metabolic regulation is associated with a wide range of chronic conditions, including obesity, insulin resistance, dyslipidemia, nonalcoholic fatty liver disease, age-related metabolic decline, and reduced tolerance to physiological stress. These conditions are increasingly understood to arise not from isolated defects in energy balance, but from coordinated dysregulation of mitochondrial function, cellular stress responses, nutrient-sensing pathways, and gut barrier integrity.

[0003] At the cellular level, metabolic dysfunction is closely linked to impaired mitochondrial energy utilization, excessive oxidative stress, chronic low-grade inflammation, and disruption of detoxification pathways. These processes contribute to loss of metabolic flexibility, accumulation of cellular damage, and reduced resilience to dietary, oxidative, and environmental stressors. Similarly, deterioration of gut barrier integrity and altered gut signaling further amplify systemic inflammation and metabolic instability, creating a reinforcing cycle that accelerates aging and functional decline.

[0004] Existing approaches to improving metabolic health frequently focus on single physiological targets such as appetite suppression, hormonal modulation, or nutrient absorption. While such strategies may produce short-term metabolic improvements, they often fail to address the underlying cellular and tissue-level processes that govern long-term metabolic resilience and stress tolerance. Moreover, interventions that act on narrow targets may be associated with undesirable side effects, limited durability of benefit, or disruption of normal homeostatic signaling.

[0005] Nutritional and dietary supplement strategies have been explored as safer, long-term alternatives for supporting metabolic health. However, many existing formulations rely on individual bioactive compounds or empirically selected combinations that do not engage multiple conserved stress-response pathways in a coordinated manner. As a result, such approachesDocket No. NEM-103-PCT Customer No. 51079 frequently yield modest, inconsistent, or incomplete benefits, particularly in complex conditions where metabolic dysfunction, oxidative stress, gut integrity, and aging processes are interdependent.

[0006] Recent advances in systems biology have highlighted the importance of integrated regulation of mitochondrial metabolism, oxidative stress defense, nutrient-sensing signaling, and intestinal barrier function in maintaining metabolic health and promoting longevity. Compositions capable of engaging these conserved biological programs simultaneously, particularly through defined combinations of bioactive compounds with complementary and synergistic activities, represent a promising but underdeveloped strategy for improving metabolic health while enhancing systemic stress resilience.

[0007] Accordingly, there exists an unmet need for compositions and methods that improve metabolic health while simultaneously strengthening cellular stress resilience, supporting gut integrity, enhancing oxidative defense, and promoting functional longevity, without reliance on appetite suppression, endocrine manipulation, or inhibition of nutrient absorption. The present invention addresses this need by providing compositions and methods that engage multiple conserved biological pathways to deliver coordinated metabolic and stress-resilience benefits.

[0008] The present invention attempts to solve these problems, as well as others.SUMMARY OF THE INVENTION

[0009] Provided herein are methods and compositions for improving the health of a subject including metabolic health, gut health, longevity, and stress resilience.

[0010] The methods and compositions are set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the methods and compositions. The advantages of the methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the methods and compositions, as claimed.

[0011] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within theDocket No. NEM-103-PCT Customer No. 51079 scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U. S. C. § 112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In the accompanying figures, like elements are identified by reference numerals among the several preferred embodiments of the present invention.

[0013] Fig. 1 is a table showing the Differential Gene Expression Summary. Human homologs were mapped to C. elegans genes using OrthoList with one-to-many mappings. Total DEGs include all genes with adj. p value <0.1 and abs(Log2FoldChange) > 0.5. Small effects on gene expression may be due to time point of collection (not long enough).

[0014] Figs. 2A-2C are Venn diagrams showing the gene expression overlap analysis of C. elegans. Fig. 2A is a graph showing the full data overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 2B is a graph showing the upregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 2C is a graph showing the downregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A.

[0015] Figs. 3A-3C are Venn diagrams showing the gene expression overlap analysis of H. sapiens. Fig.3A is a graph showing the overlap of significant genes for Fisetin, Rosmarinic Acid, and Urolithin A. Fig.3B is a graph showing the upregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 3C is a graph showing the downregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A.

[0016] Figs.4A-4B are graphs showing that NL100P3 exhibits lifespan enhancement in wild type cohort. Fig. 4C is a table showing the placebo, Urolithin A and NL100P3 treatment. ** Urolithin A 50 μM = Urolithin 11.4μg / mL.Docket No. NEM-103-PCT Customer No. 51079

[0017] Figs.5A-5B are graphs showing NL100P3 suppress stimulated neural activity in adults. ** Phosphatidyl serine 50 uM = Phosphatidyl serine 19.3 ug / mL. Enrolled worm-subjects are the same as the longevity study.

[0018] Figs.6A-6B are graphs that show NL100P3 extend lifespan under chronic metabolic stress with ** Semaglutide 2.4 uM = Semaglutide 9.87 ug / mL. Fig. 6C is a table that shows NL100P3 extend lifespan under chronic metabolic stress. Figs. 6D-6E is a graph and a table that shows the binary compounds extend lifespan under chronic metabolic stress Rosmarinic acid 100 uM + Urolithin A 100 uM, Fisetin 100 uM + Urolithin A 100 uM, and Fisetin 100 uM + Rosmarinic acid 100 uM. Fig. 6F is a table that shows the full summary of the compounds tested for the metabolic health study. Fig. 6G is a graph showing the Percent survival on day 16. FIG. 6H is a graph showing the ranking of NL100P3, NL100P2A, NL100P2B, and NL100P2C compared with the industry standard in terms of percentage change in survival on day 16.

[0019] Figs. 7A-7B are graphs that show NL100P3 confer strong oxidative stress resilience showing that NL100P3 exhibits lifespan enhancement in wild type cohort. ** Ascorbic acid 10 mM = Ascorbic acid 1.76 mg / mL = 440 mg of human dose. % changes are calculated based on day 12 data. FIG. 7C is a table that shows NL100P3 exhibits strong oxidative stress resilience.

[0020] Fig. 8A is a schematic of the conceptual integrated mechanism-of-action model for NL100P3 under standard dietary conditions. The schematic synthesizes pathway- and gene-level transcriptional evidence into a unified biological framework linking enhanced mitochondrial lipid utilization and NRF2-associated detoxification with coordinated suppression of PI3K-AKT-mTOR nutrient-sensing and growth signaling. Arrows indicate directionality of pathway engagement inferred from significantly regulated genes (|log2FC| > 0.5, adjusted P < 0.05).

[0021] Fig. 8B is a bubble plot showing the pathway-level transcriptional changes induced by NL100P3 under standard diet and the significantly enriched pathways derived from differentially expressed genes in C. elegans following NL100P3 exposure (log2 fold change > 0.5, adjusted P < 0.05). Red and blue dots indicate upregulated and downregulated pathways, respectively, and dot size represents the number of genes contributing to each pathway.

[0022] Fig. 8C is a table showing the structured mapping of NL100P3 -responsive pathways to claim-relevant signals and functional outcome domains and representing significantly regulated pathways in C. elegans following NL100P3 exposure, organized by associated benefit indications, molecular signals supporting claim construction, and functional outcome domains. UpregulatedDocket No. NEM-103-PCT Customer No. 51079 pathways (A) align with detoxification and mitochondrial lipid metabolism programs, whereas downregulated pathways (▼) correspond to nutrient-sensing and senescence-associated signaling, illustrating functional convergence across detoxification, metabolic health, and longevity-associated regulatory axes.

[0023] Fig. 8D is a graph of the Gene-level transcriptional regulation induced by NL100P3 under standard diet showing the differential expression of conserved genes in C. elegans following NL100P3 administration under standard dietary conditions. Bars represent log2 fold change for significantly regulated genes (|log2FC| > 0.5, adjusted P < 0.05), grouped by functional category: longevity-associated regulatory genes (pink), metabolic health genes (blue), and detoxification genes (green). NL100P3 induces coordinated upregulation of genes involved in mitochondrial lipid metabolism, oxidative phosphorylation, and phase I / II detoxification, alongside downregulation of nutrient-sensing and senescence-associated regulatory genes.

[0024] Fig. 8E is a table showing the Upregulated genes with conserved detoxification and metabolic functions following NL100P3 administration and the significantly upregulated genes in C. elegans following NL100P3 exposure under standard dietary conditions (|log₂FC| > 0.5, adjusted P < 0.05). Listed genes are annotated with conserved human orthologs and grouped by functional category. Upregulated markers include phase I and phase II detoxification enzymes (cytochrome P450s, UDP-glucuronosyltransferases, glutathione pathway components) and mitochondrial energy metabolism genes involved in fatty acid -oxidation and oxidative phosphorylation, indicating activation of conserved protective and metabolic programs.

[0025] Fig. 8F is a table showing the Downregulated genes associated with nutrient-sensing and senescence-related regulation following NL100P3 administration and the significantly downregulated genes in C. elegans following NL100P3 exposure under standard dietary conditions (|log₂FC| > 0.5, adjusted P < 0.05). Suppressed genes include conserved regulators of mTOR signaling (rict-l / RICTOR), transcriptional and cell-cycle control (cdk-9 / CDK9, lin-9 / LIN9 and circadian-linked growth regulation (lin-42 / PER1–3), consistent with attenuation of nutrient-sensing and pro-senescent regulatory programs.

[0026] Fig. 9A is a graph showing the lifespan analysis revealed a consistent extension of median survival in NL100P3 -treated condition with NL100P3 compared to placebo across two independent studies.Docket No. NEM-103-PCT Customer No. 51079

[0027] Fig. 10A are graphs showing the improved survival under oxidative stress conditions for NL100P3, urolithin A, rosmarinic acid, fisetin, urolithin A + fisetin, urolithin A + rosmarinic acid, rosmarinic acid + fisetin vs. control of DMSO. Fig. 10B is the table for the data in Fig. 15A. Fig.10C are graphs showing the additive survival benefits under oxidative stress with NL100P3 components.

[0028] Fig. 11A is a graph showing the metabolic health effects ofNL100P3 were evaluated using both analytical-grade compounds and supplier-grade ingredient materials. Mean and median survival under high-sugar conditions were increased relative to placebo across all material sources, demonstrating that the metabolic health benefits of NL100P3 are preserved when using commercially sourced ingredients. Fig. 11B is a graph showing NL100P3 Maintains Metabolic Health Benefits Across Dose Range and Ingredient Sources.

[0029] Fig. 12A is a graph showing the Reduction of Sugar-Induced Fat Accumulation in C. elegans. Fig. 12B is a graph showing the Consistent Reduction in Fat Accumulation Using Supplier-Grade Ingredients. ** Ascorbic acid 10 mM = Ascorbic acid 1.76 mg / mL = 440 mg of human dose. % changes are calculated based on day 12 data. FIG. 7C is a table that shows NL100P3 extends lifespan under chronic metabolic stress compared to semaglutide.

[0030] Fig. 13A is a graph showing NL100P3 enhances intestinal barrier integrity. Fig. 13B is a graph showing the intestinal lumen distention is reduced by NL100P3 Treatment in the anterior intestine. Fig. 13B is a graph showing the intestinal lumen distention is reduced by NL100P3 Treatment in the posterior intestine.DETAILED DESCRIPTION OF THE INVENTION

[0031] The foregoing and other features and advantages of the invention are apparent from the following detailed description of exemplary embodiments, read in conjunction with the accompanying drawings. The detailed description and drawings are merely illustrative of the invention rather than limiting, the scope of the invention being defined by the appended claims and equivalents thereof.

[0032] Embodiments of the invention will now be described with reference to the Figures, wherein like numerals reflect like elements throughout. The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive way, simply because it is being utilized in conjunction with detailed description of certain specific embodiments of the invention. Furthermore, embodiments of the invention may include several novel features, no single one ofDocket No. NEM-103-PCT Customer No. 51079 which is solely responsible for its desirable attributes or which is essential to practicing the invention described herein.

[0033] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. It will be further understood that the terms “comprises,” “comprising,” “includes,” and / or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0034] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. The word “about,” when accompanying a numerical value, is to be construed as indicating a deviation of up to and inclusive of 10% from the stated numerical value. The use of any and all examples, or exemplary language (“e.g.” or “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the invention.

[0035] References to “one embodiment,” “an embodiment,” “example embodiment,” “various embodiments,” etc., may indicate that the embodiment(s) of the invention so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment,” or “in an exemplary embodiment,” do not necessarily refer to the same embodiment, although they may.

[0036] As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to beDocket No. NEM-103-PCT Customer No. 51079 limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0037] Definitions

[0038] The term “administration” or “administering” refers to a method of providing a dosage of a compound or active ingredient or pharmaceutical composition to a subject, where the method is epicutaneous (topical) or subcutaneous. Modes of administration, dosing schedules disclosed compounds and compositions can be determined according to the criteria generally taken into account in the establishment of a metabolic disorder treatment adapted to, for example, a patient's metabolic pattern. The compositions can be administered such that they conditioned to be treated before a metabolic disorder occurs.

[0039] In the present disclosure, an “effective amount” or an “effective dose” of the composition, or composition refers to an amount of composition that, once administered to a subject, will reach the subject’s bloodstream and / or bodily tissues and achieve the effect disclosed herein. It will be understood, however, that the total daily usage may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of ergothioneine employed; the duration of the treatment; drugs used in combination or coincidental with active compounds disclosed herein; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. In addition, a “therapeutically effective amount” is the amount that will elicit the biological or medical response of a tissue, system, or subject that is being sought by a researcher or clinician, and in particular elicit some desired therapeutic or prophylactic effect for extending lifespan or improving longevity in a mammal. The term effective amount may include or be synonymous with a pharmaceutically effective amount or a therapeutically effective amount.Docket No. NEM-103-PCT Customer No. 51079

[0040] The term “composition” as used herein has its conventional meaning and refers to a composition which is consumable or administered by a subject. Compositions, or they may be formulated as foods (including drinks) or dietary supplements. They may also be formulated as powders that are admixed with a food or beverage immediately before administration or consumption. The term “pharmaceutically acceptable” as used herein has its conventional meaning and refers to compounds, material, compositions and / or dosage forms, which are, within the scope of sound medical judgment suitable for contact with the tissues of mammals, especially humans, without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio. Composition includes configurational isomers (such as cis and trans isomers) and all optical isomers (such as enantiomers) Isomers and diastereomers), racemic, diastereoisomers and other mixtures of these isomers, as well as solvates, hydrates, isomorphs, polymorphs, tautomers, ester, salt forms and prodrugs. The term “prodrug” refers to a compound that is a drug precursor, which releases the drug in vivo through some chemical or physiological processes after administration (for example, the prodrug is transformed into the desired drug form when it reaches physiological pH or through the action of enzymes). Exemplary prodrugs release the corresponding free acid upon cleavage, and the hydrolyzable ester-forming residues of the compounds of the present invention.

[0041] The term “pharmaceutically acceptable” refers to derivatives, analogues and salts which are physiologically acceptable for use in mammals, and which are not unduly toxic or otherwise unacceptable for such use. The term “mammals” includes human and non-human mammals, including domestic animals, e.g. cats, dogs, rodents, cattle, horses and the like, as well as nondomesticated animals.

[0042] The term “synergistic” as used herein is refers to the phenomenon wherein the cumulative pharmacological effect of two or more ingredients when used in combination is higher than the sum of the effect of each of them tested individually. The term “potentiating” as used herein refers to the phenomenon where the efficacy of an active ingredient is significantly enhanced when it is combined with a second ingredient, wherein said second ingredient itself does not demonstrate any efficacy in the same pharmacological test. In some cases of potentiation, not only is said second ingredient devoid of the pharmacological effect being measured, it may even cause an opposite effect, when assayed alone. An example of such a case would be as follows: ingredient A is anti-inflammatory; ingredient B is pro-inflammatory; when A and B are combined, saidDocket No. NEM-103-PCT Customer No. 51079 combination produces an anti-inflammatory effect that is greater than seen with A alone. In the context of the present invention, potentiation is regarded as a special case of synergism. Thus, the term ‘synergism’ (or synergistic, or the like), when used to define the properties of a composition of the present invention, also includes within its range of meaning the potentiation effect described immediately hereinabove.

[0043] The term “mammal” or “subject” may be used interchangeably to refer to any animal to which the presently disclosed methods and compositions may be applied or administered. The animal may have an illness or other disease, but the animal does not need to be sick to benefit from the presently disclosed methods and compositions. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cattle, horses, primates, such as monkeys, chimpanzees, and apes, and, in particular, humans.

[0044] The term “metabolic health” or “metabolic stress” may related to disease or condition selected from the group consisting of metabolic syndrome, reduced metabolic rate, metabolic stress, cardiovascular disease, endothelial cell dysfunction, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis (NASH), drug-induced liver injury, drug-induced cravings, anaemia disorders, al -antitrypsin deficiency, ischemia / reperfusion injury, inflammation, aging of the skin, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, anxiety disorder, ulceration, amyotrophic lateral sclerosis, age-related macular degeneration, mitochondrial diseases, (including for example poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction sometimes learning disabilities, and dementia as a result of mitochondrial disease. Further diseases related to mitochondrial dysfunction include: Diabetes mellitus and deafness (DAD); Leber's hereditary optic neuropathy (LHON); Leigh syndrome (subacute sclerosing encephalopathy); neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); myoneurogenic gastrointestinal encephalopathy (MNGIE); Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS); and mtDNA depletion), sporadic inclusion body myositis (sIBM), cancer, cognitive disorder, stress, and mood disorder; for improving cognitive function;Docket No. NEM-103-PCT Customer No. 51079 for weight management; or to increase muscle or mental performance. The compounds of formula (I) or (la) or salts thereof are particularly suitable for use in improving muscle function, muscle strength, muscle endurance and muscle recovery.

[0045] The term “improving”, “enhancing” or “treating” are used interchangeably herein. These terms can refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit can mean eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0046] The method and composition may be used in the treatment of a disease or condition selected from the group consisting of metabolic syndrome, reduced metabolic rate, metabolic stress, cardiovascular disease, endothelial cell dysfunction, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, drug-induced cravings, anaemia disorders, al -antitrypsin deficiency, ischemia / reperfusion injury, inflammation, aging of the skin, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, anxiety disorder, ulceration, amyotrophic lateral sclerosis, age-related macular degeneration, cancer, cognitive disorder, stress, and mood disorder; for improving cognitive function; for weight management; or to increase muscle or mental performance.

[0047] Description of Embodiments

[0048] Generally speaking, the invention is a method and composition comprising an effective amount of active compounds improving metabolic health, improving longevity, improving fat reduction, improving gut health, and / or stress resilience in a mammal. The method andDocket No. NEM-103-PCT Customer No. 51079 composition comprises a formulation of binary and tertiary combinations of Fisetin, Rosmarinic acid, and Urolithin A. In one embodiment, the method and composition comprise an effective amount of Fisetin and an effective amount of Urolithin A (hereinafter NL100P2A). In another embodiment, the method and composition comprise an effective amount of Urolithin A and an effective amount of Rosmarinic acid (hereinafter NL100P2B). In another embodiment, the method and composition comprises an effective amount of Rosmarinic acid and an effective amount of Fisetin (hereinafter NL100P2C). In another embodiment, the method and composition comprise an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A (hereinafter NL100P3). NL100P2A, NL100P2B, NL100P2C, and NL100P3 all show synergistic effects and improve metabolic health significantly. In addition, NL100P3 also improves stress resilience, survival, and gut health significantly.

[0049] The combination of an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A enhances their collective efficacy and synergistically provides antioxidant defense by protects cells from oxidative stress. The combination of an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A enhances their collective efficacy and synergistically provides inflammation modulation by mitigating inflammation in the gut, metabolic systems, and stress responses. The combination of an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A enhances their collective efficacy and synergistically provides mitochondrial and cellular health by promoting energy efficiency and longevity through autophagy and senolysis. The combination of an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A enhances their collective efficacy and synergistically provides gut integrity and microbiota balance by strengthening gut health for overall systemic benefits.

[0050] In one embodiment, an effective amount of Fisetin and an effective amount of Urolithin A is at a ratio of at least 1: 1 or any fraction from 1:99 or 99: 1 for improving metabolic health, stress resilience, gut health, cognitive calmness, and longevity. In one embodiment, an effective amount of Urolithin A and an effective amount of Rosmarinic acid is at a ratio of at least 1:1, or any fraction from 1:99 or 99:1 for improving metabolic health, stress resilience, gut health, cognitive calmness, and longevity. In another embodiment, an effective amount of Rosmarinic acid and an effective amount of Fisetin is at a ratio of at least 1:1 or any fraction from 1:99 or 99:1 forDocket No. NEM-103-PCT Customer No. 51079 improving metabolic health, stress resilience, gut health, cognitive calmness, and longevity. In another embodiment, an effective amount of Fisetin, an effective amount of Rosmarinic acid and an effective amount of Urolithin A is at a ratio of at least 1:1:1 or any fraction from 1: 1:99, 1:99: 1, 99: 1:99, 1:99:99, or 99: 1: 1 for improving metabolic health, stress resilience, gut health, cognitive calmness, and longevity. In one embodiment, the method and composition further comprise improving gut health under metabolic stress conditions of the subject. The method and composition provide gut health benefits under a high sugar diet, as supported by the examples below.

[0051] In one embodiment, the method and composition do not work through the GLP-1 Receptor Agonists or Dual GLP-1 and GIP receptor agonist or blocking fat absorption in the digestive tract and hence does not show those side effects of GLP-1 receptor agonists. On the contrary, the method and composition enhances longevity by about 16 %, shown comparable neuromuscular health as control. As used herein, “enhances longevity” or “improving longevity” refers to the increase of the length of time for which a mammal lives. An example of improving longevity can be seen in Examples below.

[0052] In one embodiment, the method and composition induce broad stimulation of canonical signal transduction pathways under high sugar diet, including receptor Tyrosine kinase, WNT, Hedgehog, NOTCH, TGF-beta family, mTOR and Rho GTPase signaling cascades. In one embodiment, Fisetin and Rosmarinic acid deliver stress resilience, metabolic and gut health benefits. In one embodiment, Urolithin A is designed to prevent the disproportionate loss of lean mass relative to fat and maintain muscle health during rapid or significant weight loss. In one embodiment, Fisetin, Rosmarinic acid, and Urolithin A are formulated to enhance metabolic health.

[0053] In one embodiment, the maximum dosage of each individual ingredient in any combination does not exceed 100 pM, or dosed as indicated below. In one embodiment, the compositions NL100P2A, NL100P2B, NL100P2C, and NL100P3 are formulated for oral administration, or formulated as detailed below. In one embodiment, the oral formulation is a dietary supplement.

[0054] Fisetin

[0055] Fisetin is a flavonoid, a group of plant pigments known for their antioxidant and antiinflammatory properties. Fisetin is characterized by multiple hydroxyl groups attached to its flavonoid structure. The chemical structure of Fisetin is shown by formula (1):Docket No. NEM-103-PCT Customer No. 51079

[0056] (1).

[0057] Fisetin has known human metabolites that include (2S,3S,4S,5R)-6-[2-(3,4-dihydroxyphenyl)-7-hydroxy-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid -a complex organic molecule, specifically a derivative of ascorbic acid (vitamin C) with a substituted chromone ring attached at the 6-position is likely to have potential medicinal uses related to its antioxidant, anti-inflammatory, and potentially anti-cancer properties, due to the presence of multiple phenolic groups and its flavonoid-like structure.

[0058] Fisetin scavenges free radicals and suppresses inflammation by inhibiting NF-KB activation and cytokine production (TEAC assay, NF-KB inhibition). Fisetin modulates gut microbiota and reduces gut inflammation in models of inflammatory bowel disease (IBD). Fisetin improves metabolic health markers like liver function and insulin sensitivity in animal models. Fisetin is shown to increase healthspan and lifespan in various animal studies.

[0059] Fisetin reduces oxidative stress and inflammation, enhances liver function and reduces insulin resistance, and promotes healthy aging by targeting cellular senescence. Fisetin modulates gut microbiota and reduces gut lining inflammation, restores beneficial bacteria and mitigates colitis symptoms, and decreases senescent cell accumulation in the gut lining. Fisetin reduces mitochondrial dysfunction and supports antioxidant defenses and promotes overall cellular health and energy metabolism through mitochondrial protection.

[0060] Fisetin derivatives may also be included, as well as prodrugs, analogues and salts. Fisetin derivatives are modified versions of fisetin, a dietary flavonol, that have been created to improve its biological properties. Fisetin derivatives include: 7-OH-fisetin, 2,2'-((4-(3,7-bis(carboxymethoxy)-4-oxo-4H-chromen-2-yl)-l,2-phenylene)bis(oxy))diacetic acid, 3, 7,3', 4'-tetra-O-acetylfisetin, 3,7,3',4'-tetrahydroxyflavone, (4-((7 -acetoxy -2-(2,2-Docket No. NEM-103-PCT Customer No. 51079 diphenylbenzo[d][l,3]dioxol-5-yl)-4-oxo-4H-chromen-3-yl)oxy)butyl) triphenylphosphonium iodide (mF3) or (4-((3,5-diacetoxy-2-(2,2-diphenylbenzo[d][l,3]dioxol-5-yl)-4-oxo-4H-chromen-7-yl)oxy)butyl) triphenylphosphonium iodide (mF7).

[0061] Rosmarinic acid

[0062] Rosmarinic acid is a phenolic acid, which are a subclass of polyphenols with strong antioxidant and anti-inflammatory activity. Rosmarinic acid is an ester formed from caffeic acid and 3,4-dihydroxyphenyllactic acid, commonly found in plants of the Lamiaceae family. Rosmarinic acid has the chemical structure according to formula (2):

[0063] (2).

[0064] Rosmarinic acid inhibits lipid peroxidation and modulates immune response. Rosmarinic acid supports gut health by improving intestinal flora and gut barrier integrity. Rosmarinic acid has potential therapeutic applications for metabolic diseases, IBD, and neurodegenerative conditions. Rosmarinic acid has efficacy in managing dementia and cognitive impairment through clinical trials. Rosmarinic acid is shown to improve insulin sensitivity and reduce metabolic disease risk by regulating glucose metabolism. Rosmarinic acid inhibits lipid peroxidation and neutralizes reactive oxygen species (ROS).

[0065] Rosmarinic acid improves insulin sensitivity in skeletal muscle, reduces risks of metabolic diseases, including diabetes and neurodegenerative conditions. Rosmarinic acid strengthens the intestinal barrier, preventing leaky gut syndrome by reducing permeability, and regulates gut flora and reduces inflammatory damage. Rosmarinic acid modulates oxidative stress and supports the immune system and has the benefits in managing cognitive decline and improving mental performance. Rosmarinic acid suppresses prostacyclin and inflammatory mediators.Docket No. NEM-103-PCT Customer No. 51079

[0066] Rosmarinic acid derivatives may also be included, as well as prodrugs, analogues and salts. Rosmarinic acid (RA) derivatives are molecules that are chemically or naturally derived from RA, a bioactive compound found in plants. RA derivatives have been shown to have many biological activities, including anti-inflammatory, antioxidant, and anti-microbial properties. Lithospermic acid A: A popular RA derivative that is a compound of RA and caffeic acid. Lithospermic acid B: A more complex RA derivative that is an ester of caffeine. Eritrichin: A trimer of caffeic acid isolated from Eritrichium sericeum. Salvianolic acid K: An RA derivative isolated from a plant. Melitric acid A: An RA derivative isolated from a plant. Rosmarinic acid methyl ester: A chemically synthesized RA derivative that has been isolated as a natural antimicrobial agent. Propyl ester of rosmarinic acid: A chemically synthesized RA derivative that has been isolated as a natural antimicrobial agent. Hexyl ester of rosmarinic acid: A chemically synthesized RA derivative that has been isolated as a natural antimicrobial agent. Biological activities of RA and its derivatives.

[0067] Urolithin A

[0068] Urolithin A belongs to the class of organic compounds called benzo-coumarins or dibenzo-a-pyrones. It is a metabolite produced by gut bacteria when they break down ellagitannins (Ellagitannins (ET) and ellagic acid (EA) are polyphenols), a type of polyphenol found in foods like pomegranates, berries, and walnuts. Urolithin A has the chemical structure according to formula (3):,, XXJ

[0069] (3).

[0070] Urolithin A has the benefits for muscle health by promoting mitochondrial function and autophagy. Urolithin A activates AMPK, promoting energy production and autophagy. Urolithin A inhibits mTOR, reducing oxidative stress and enhancing mitochondrial quality. Urolithin ADocket No. NEM-103-PCT Customer No. 51079 strengthens the intestinal barrier and reduces inflammatory responses and suppresses pro-inflammatory cytokines and oxidative stress. Urolithin A demonstrated benefits in muscle health, glucose metabolism, and aging-related conditions.

[0071] Urolithin A activates AMPK to enhance energy production, inhibits mTOR, promoting autophagy and reducing oxidative stress, and improves glucose metabolism and supports mitochondrial health. Urolithin A enhances gut microbiota diversity and strengthens the intestinal barrier and reduces gut inflammation and leakage of harmful substances. Urolithin A protects cells from mitochondrial dysfunction and supports cellular maintenance and energy balance during stress and aging.

[0072] Urolithin A derivatives may also be included, as well as prodrugs, analogues and salts. Urolithin A (UA) derivatives are compounds that are chemically modified versions of urolithin A, including 6H-dibenzo[b,d]pyran-6-one derivatives, that is, 6H-benzo[c]chromen-6-ones, with different hydroxyl substitutions. They are created to improve the bioavailability of UA, which is a gut metabolite that has many potential health benefits. UA derivatives are created by conjugating UA with nonsteroidal anti-inflammatory drugs (NSAIDs) like ibuprofen, aspirin, and diclofenac. UA derivatives are created by methylating the phenolic hydroxyl groups of UA with methyl iodide and K2CO3. UA derivatives are created by demethylating the phenolic methyl ether in UA with AlCl₃. UA derivatives can improve the bioavailability of UA, which is limited by phase II intestinal metabolism. UA derivatives can have anti-inflammatory properties, which may help with inflammation-related conditions. UA derivatives can have inhibitory activity against phosphodiesterase II (PDE2).

[0073] Dosage

[0074] In one embodiment, the weight ratio of Fisetin + Urolithin A is about 1.0: 1.0, Urolithin A + Rosmarinic is about 1.0: 1.0, Rosmarinic acid + Fisetin is about 1.0: 1.0, Fisetin + Rosmarinic acid + Urolithin A is about 1.0: 1.0: 1.0. In other embodiments, the weight ratio of Fisetin + Urolithin A ranges from 1.0:2.0 to 2.0: 1.0, Urolithin A + Rosmarinic ranges from 1.0:2.0 to 2.0: 1.0, Rosmarinic acid + Fisetin ranges from 1.0:2.0 to 2.0: 1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:2.0: 1.0 to 2.0: 1.0: 1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:2.0:2.0 to 2.0:2.0:1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:2.0:1.0 to 2.0:1.0:2.0, and Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:2.0:2.0 to 1.0:2.0:1.0. In additional embodiments of the forgoing, the weight ratio of Fisetin + Urolithin A ranges from 1:0.9Docket No. NEM-103-PCT Customer No. 51079 to 0.9 to 1, Urolithin A + Rosmarinic ranges from 1:0.9 to 0.9 to 1, Rosmarinic acid + Fisetin ranges from 1:0.9 to 0.9:1, Fisetin + Rosmarinic acid + Urolithin A ranges from 1:0.9:0.9 to 0.9:0.9:1, Fisetin + Rosmarinic acid + Urolithin A ranges from 1:0.9: 1.0 to 0.9: 1.0: 1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 0.9: 1:0.9 to 0.9:0.9:1, and Fisetin + Rosmarinic acid + Urolithin A from 0.9: 1:0.9 to 0.9: 1.0: 1.0. In additional embodiments of the forgoing, the weight ratio of Fisetin + Urolithin A ranges from 1:0.8 to 0.8 to 1, Urolithin A + Rosmarinic ranges from 1:0.8 to 0.8 to 1, Rosmarinic acid + Fisetin ranges from 1:0.8 to 0.8 to 1, Fisetin + Rosmarinic acid + Urolithin A ranges from 1:0.8:0.8 to 0.8:0.8: 1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1:0.8: 1.0 to 0.8:0.8: 1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:0.8:0.8 to 0.8: 1.0:0.8, and Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0: 1.0:0.8 to 0.8:0.8: 1.0. In a specific embodiment, the weight ratio of Fisetin + Urolithin A ranges from 1.2: 1.0 to 1.0: 1.2, Urolithin A + Rosmarinic acid ranges from 1.2: 1.0 to 1.0: 1.2, Rosmarinic acid + Fisetin ranges from 1.2: 1.0 to 1.0: 1.2, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.2: 1.2: 1.0 to 1.0: 1.2: 1.2, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.2: 1.0: 1.2 to 1.0: 1.2: 1.2, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0: 1.2: 1.0 to 1.2: 1.0: 1.2, and Fisetin + Rosmarinic acid + Urolithin A ranges from 1.2: 1.0: 1.2 to 1.0: 1.2: 1.0. In a specific embodiment, the weight ratio of Fisetin + Urolithin A ranges from 1.1: 1.0 to 1.0: 1.1, Urolithin A + Rosmarinic acid ranges from 1.1:1.0 to 1:0:1.1, Rosmarinic acid + Fisetin ranges from 1.1:1.0 to 1.0:1.1, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.1:1.1:1.0 to 1.0:1.1:1.1, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.1: 1.1: 1.0 to 1.0:1.1:1.0, Fisetin + Rosmarinic acid + Urolithin A ranges from 1.1: 1.0: 1.1 to 1.0: 1.1: 1.0, and Fisetin + Rosmarinic acid + Urolithin A ranges from 1.0:1.0:1.1 to 1.0:1.1:1.1.

[0075] In some embodiments, the method and composition provided herein may include about 0.1 pM to 100 pM of Fisetin per effective dose or effective amount. For example, a composition provided herein may comprise between 0.1 pM to 90 pM, 0.5 pM to 90 pM, 1 pM to 90 pM, 0.1 pM to 95 pM, 0.5 pM to 95 pM, 1 pM to 95 pM, 0.1 pM to 85 pM, 0.5 pM to 85 pM, 1 pM to 85 pM, 0.1 pM to 80 pM, 0.5 pM to 80 pM, 1 pM to 80 pM, 0.1 pM to 75 pM, 0.5 pM to 75 pM, 1 pM to 75 pM, 0.1 pM to 99 pM, 0.5 pM to 99 pM, 1 pM to 99 pM, 0.1 pM to 70 pM, 0.5 pM to 70 pM, 1 pM to 70 pM, 2 pM to 95 pM, 2 pM to 90 pM, 2 pM to 85 pM, 2 pM to 99 pM, 2 pM to 80 pM, 2 pM to 75 pM, or between 2 pM to 95 pM of Fisetin per effective dose. Alternatively, the dose may comprise 10 pM to about 100 pM of Fisetin per effective dose.Docket No. NEM-103-PCT Customer No. 51079

[0076] In some embodiments, the method and composition provided herein may include about 0.1 pM to 100 pM of Rosmarinic acid per effective dose or effective amount. For example, a composition provided herein may comprise between 0.1 pM to 90 pM, 0.5 pM to 90 pM, 1 pM to 90 pM, 0.1 pM to 95 pM, 0.5 pM to 95 pM, 1 pM to 95 pM, 0.1 pM to 85 pM, 0.5 pM to 85 pM, 1 μM to 85 μM, 0.1 μM to 80 μM, 0.5 μM to 80 μM, 1 μM to 80 μM, 0.1 μM to 75 μM, 0.5 μM to 75 μM, 1 μM to 75 μM, 0.1 μM to 99 μM, 0.5 μM to 99 μM, 1 μM to 99 μM, 0.1 μM to 70 μM, 0.5 μM to 70 μM, 1 μM to 70 μM, 2 μM to 95 μM, 2 μM to 90 μM, 2 μM to 85 μM, 2 μM to 99 μM, 2 μM to 80 μM, 2 μM to 75 μM, or between 2 μM to 95 μM of Rosmarinic acid per effective dose. Alternatively, the dose may comprise 10 pM to about 100 pM of Rosmarinic acid per effective dose.

[0077] In some embodiments, the method and composition provided herein may include about 0.1 pM to 100 pM of R Urolithin A per effective dose or effective amount. For example, a composition provided herein may comprise between 0.1 pM to 90 pM, 0.5 pM to 90 pM, 1 pM to 90 pM, 0.1 pM to 95 pM, 0.5 pM to 95 pM, 1 pM to 95 pM, 0.1 pM to 85 pM, 0.5 pM to 85 pM, 1 pM to 85 pM, 0.1 pMto 80 pM, 0.5 pMto 80 pM, 1 pMto 80 pM, 0.1 pMto 75 pM, 0.5 pMto 75 pM, 1 pM to 75 pM, 0.1 pM to 99 pM, 0.5 pM to 99 pM, 1 pM to 99 pM, 0.1 pM to 70 pM, 0.5 pM to 70 pM, 1 pM to 70 pM, 2 pM to 95 pM, 2 pM to 90 pM, 2 pM to 85 pM, 2 pM to 99 pM, 2 pM to 80 pM, 2 pM to 75 pM, or between 2 pM to 95 pM of Urolithin A per effective dose. Alternatively, the dose may comprise 10 pM to about 100 pM of Urolithin A per effective dose.

[0078] The optimal dosage will depend upon the intended use of the composition, and upon the individual subject. Dosing may for example be daily to weekly. In one embodiment, dosing is at least weekly. For example, a subject may receive one dose once weekly, twice weekly, thrice weekly, or every other day. In one embodiment, dosing is at least daily. For example, a subject may receive one or more doses daily. It is believed that dosing for greatest efficacy in humans involves extended, daily administration. Where extended use is contemplated, this may include use for 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or even longer.

[0079] In certain embodiments, the dosage form is formulated as granules, pellets, micro particles, tablet, hard shell capsules, suspended in a liquid, suspended in a syrup or enema. In certain embodiments, the dosage form is formulated for oral or mucosal delivery. In certain embodiments, the dosage form is formulated as or in a lozenge, candy, toffee, chocolate or cookie. In certainDocket No. NEM-103-PCT Customer No. 51079 embodiments, the tablet or pellets are an immediate release or slow or controlled release dosage forms. In certain embodiments, the tablet is a melt or dissolved in the mouth or is muco-adhesive dosage form.

[0080] In certain embodiments, the unit dosage form which is a unit particles, such as tablet, capsule, granules, pellets, micro-particles and film, are enteric coated or coated with a colonic coat that protect the unit dose from being decomposed at the acidic gastric pH and swells in time manner of pH controlled manner or both, to release the composition at the distal intestine and may also release part of the composition in the intestines for systemic absorption and part of the composition for pharmacological effect.

[0081] In certain embodiments, the composition is formulated in a semi solid or liquid dosage form such as cream, lotion, ointment, dispersion, suspension, gel, foam, spray, syrup, liquid, eye drops, ear drops, enema or an oral dosage form or a topical dosage form or a local ophthalmic or optic or oral cavity or vaginal or rectal or uterine dosage form.

[0082] In certain embodiments, any one of the compositions described above, or any one of the dosage forms described above, is for use in a method of treating metabolic disorders or improving metabolic quality. Preferred dosage forms include, but are not limited to, any liquid or semi solid or solid dosage form. The composition may be formulated in a medicament by preparing a topical or mucosal or oral delivery system. The topical delivery system may be in form of eye drops, a suspension, ointment, cream, foam, spray, topical patch. The oral delivery system may be a tablet or capsule or soft capsule or sachet or granules or a syrup. The mucosal delivery system may be a gel, pessary, enema, douche, wash, foam, mucoadhesive gel or tablet for immediate or for slow or controlled release. The vehicle may comprise any acceptable solvent and inactive ingredients as well as preservatives, anti-oxidants, and coloring agents. The delivery form may be single dose or multiple doses, as well as micro particle granulate nanoparticle microcapsule liposome micelle, and the like as known in the art of pharmaceutical, cosmetic, veterinary medicine and art of formulation. Further details of suitable dosage forms may be obtained from any standard reference work in this field, including, for example: Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa, USA (1980).

[0083] Thus, in some embodiments of the present invention, the composition further comprises one or more excipients selected from the group consisting of solvents, stabilizers, suspending agents, emulsifiers, release modifying, targeting and viscosity agents and combinations thereof.Docket No. NEM-103-PCT Customer No. 51079

[0084] In some embodiments, the composition of the present invention is formulated as a dosage form selected from the group consisting of a liquid, a suspension, an emulsion, a foam, a spray, a liposome, a semi-solid, a cream, an ointment, a patch, a particulate formulation, a granulate, a micro-particulate formulation, a nano-particulate formulation, a solid dosage form, a tablet, a capsule, an orally-disintegrable capsule, a mouth wash and an adhesive buccal tablet.

[0085] According to particular embodiments, the compounds or derivatives prepared according to embodiments of the methods of the present disclosure can comprise compounds or derivatives, or salts, hydrates, solvates, or prodrugs thereof, or crystalline forms thereof, substantially free of solvents or other by-products, generally, or a particular solvent or by-product. In certain embodiments, by “substantially free” is meant greater than about 80% free of solvents or byproducts, or greater than about 80% free of a particular solvent or by-product, more preferably greater than about 90% free of solvents or by-products, or greater than about 90% free of a particular solvent or by-product, even more preferably greater than about 95% free of solvents or by-products, or greater than about 95% free of a particular solvent or by-product, even more preferably greater than 98% free of solvents or by-products, or greater than about 98% free of a particular solvent or by-product, even more preferably greater than about 99% free of solvents or by-products, or greater than about 99% free of a particular solvent or by-product, even more preferably greater than about 99.99% free of solvents or by-products, or greater than about 99.99% free of a particular solvent or by-product, and most preferably quantitatively free of solvents or by-products, or quantitatively free of a particular solvent or by-product.

[0086] For preparing pharmaceutical compositions from a hydrate, solvate, or prodrug thereof, prepared according to the methods of the present disclosure, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances that may also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.

[0087] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active components. In tablets, the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.

[0088] The powders and tablets preferably contain from about 1 to about 99.99 percent of the active form of composition, or salt, hydrate, solvate, or prodrug thereof, prepared according to theDocket No. NEM-103-PCT Customer No. 51079 methods of the present disclosure. Suitable carriers are microcrystalline cellulose, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like, and other excipients may include magnesium stearate, stearic acid, talc, silicon dioxide, etc. Dosages of the active form of composition may be between about Img to about lOOOmg in the preparation for example. The term “preparation” is intended to include the formulation of active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it. Tablets, powders, capsules, pills, sachets, and lozenges are included. Tablets, powders, capsules, pills, sachets, and lozenges can be used as solid forms suitable for oral administration. Liquid preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions. For example, parenteral injection liquid preparations can be formulated as solutions in aqueous polyethylene glycol solution. The crystalline forms of composition extracts, or salts, hydrates, solvates, or prodrugs thereof prepared according to the methods of the present disclosure may thus be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose for example in ampoules, pre-filled syringes, small volume infusion, or in multi-dose containers with an added preservative). The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. The method of administration may be via inhalation and topical routes. Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0089] Compositions suitable for topical administration include the active ingredient in an inert base such as gelatin and glycerine or sucrose and acacia; and mouthwashes comprising the active ingredient in suitable liquid carrier.Docket No. NEM-103-PCT Customer No. 51079

[0090] Solutions or suspensions are applied directly to the skin by conventional means, for example with a dropper, pipette, or spray. The compositions may be provided in single or multidose form. In compositions intended for administration to the respiratory tract, including intranasal compositions, the compound or derivative will generally have a small particle size, for example on the order of 5 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.

[0091] The pharmaceutical preparations are preferably in unit dosage forms. In such form, the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packaged tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0092] Tablets, capsules, tinctures, and lozenges for oral administration and liquids for oral use are preferred compositions. Solutions or suspensions for application to the nasal cavity or to the respiratory tract are preferred compositions. Transdermal patches for topical administration to the epidermis are preferred compositions.

[0093] Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well-known suspending agents.

[0094] Further details on techniques for formulation may be found in the latest edition of Remington’s Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).

[0095] C. elegans is a transparent worm about 1 mm in length that lives in temperate soil environments. It has been extensively used as a model organism because it has all the physiological properties of an animal, the ability to replicate human diseases, and a fast life cycle. A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms. Model organisms are widely used to research human disease when human experimentation would be unfeasible or unethical. This strategy is made possible by the common descent of all living organisms, and the conservation of metabolic and developmentalDocket No. NEM-103-PCT Customer No. 51079 pathways and genetic material over the course of evolution. C. elegans was used in the examples provided herein because it has advantages including having all the physiological properties of an animal, the ability to replicate human diseases, includes genetic homologs to human genes, and a fast life cycle. Approximately 60-80% of human protein-coding genes have C. elegans orthologs, with about 83% of the C. elegans proteome sharing homology with human proteins. Key orthologous genes are highly conserved in lipid metabolism, signaling pathways (e.g., insulin), and over 50% of human disease genes.

[0096] Pathway -lev el transcriptional reprogramming induced by NL100P3

[0097] To define the primary biological programs modulated by NL100P3 under standard dietary conditions, differentially expressed genes (DEGs) were identified in C. elegans, as detailed below. Identified DEGs were subsequently mapped to human orthologs using OrthoList2, followed by pathway enrichment analysis across multiple annotation frameworks, including Gene Ontology (biological process, molecular function, and cellular component) and curated pathway databases such as KEGG, Reactome, MSigDB, and WikiPathways. This integrative enrichment approach revealed a coordinated and directionally consistent transcriptional reprogramming affecting metabolic, detoxification, and nutrient-sensing pathways.

[0098] Among the most prominently upregulated pathways were those associated with cellular detoxification and redox metabolism, including biological oxidations, metabolism of xenobiotics by cytochrome P450 enzymes, drug metabolism, glucuronidation, and glutathione conjugation processes. Enrichment of the NRF2 pathway, together with glutathione metabolic and transferase activities, indicates activation of conserved phase I and phase II detoxification programs involved in oxidative stress handling and xenobiotic clearance.

[0099] In parallel, NL100P3 exposure resulted in significant upregulation of lipid and mitochondrial energy metabolism pathways, including metabolism of lipids, fatty acid metabolism, long-chain fatty acid metabolic processes, and mitochondrial fatty acid P-oxidation. These lipid catabolic programs were accompanied by enrichment of PPAR signaling and oxidative phosphorylation, collectively indicating enhanced mitochondrial energy utilization and transcriptional programs associated with metabolic efficiency.

[0100] Conversely, pathways linked to nutrient-sensing and longevity-limiting signaling were consistently downregulated. These included PI3K-Akt signaling, insulin resistance-associated pathways, cellular senescence-related programs, and mTOR signaling. The coordinatedDocket No. NEM-103-PCT Customer No. 51079 suppression of these pathways suggests attenuation of anabolic and pro-senescent signaling states that are widely implicated in aging biology.

[0101] Taken together, the pathway-level transcriptional profile induced by NL100P3 demonstrates a coherent biological shift characterized by activation of mitochondrial lipid utilization and detoxification programs alongside restraint of nutrient-sensing and senescence-associated pathways. This integrated pattern establishes the mechanistic foundation for a metabolically efficient and stress-resilient cellular state in C. elegans, forming the primary evidence base for subsequent gene-level, functional, and aging-hallmark analyses.

[0102] Gene-Level Molecular Substantiation of NL100P3-Induced Transcriptional Reprogramming

[0103] To substantiate the pathway-level transcriptional reprogramming induced by NL100P3 at the molecular level, individual DEGs were examined based on directionality, functional annotation, and conservation between C. elegans and H. sapiens. Gene-level changes were organized into functional classes corresponding to detoxification, metabolic health, and longevity-associated regulatory processes.

[0104] Induction of detoxification-associated gene programs

[0105] NL100P3 exposure resulted in robust upregulation of multiple genes encoding enzymes involved in conserved phase I and phase II detoxification pathways. These included cyp-14A5 (human orthologs: CYP2D6 / 7), which mediates oxidative metabolism of drugs and xenobiotics, and cyp-13A5 (human orthologs: CYP4B1 / A22), associated xenobiotic processing. Phase II conjugative detoxification was supported by induction of ugt-6 (human orthologs: UGT3A1 / 2) and ugt-62 (human orthologs: UGT2B10 / 28), which participate in glucuronidation reactions important for bile acid, amine, and drug clearance.

[0106] Consistent with redox homeostasis and cytoprotective responses, gst-1 (human orthologs: GSTP1, GSTM1 / 2 / 3 / 4 / 5) was upregulated, reflecting enhanced glutathione-dependent detoxification of reactive intermediates. Additionally, induction of ftn-2 (human orthologs: FTL, FTH1), a ferritin heavy / light chain homolog and known NRF2 target, supports engagement of iron homeostasis and oxidative stress buffering mechanisms. Collectively, these gene-level changes indicate activation of conserved detoxification and antioxidant defense programs in C. elegans.

[0107] Activation of Mitochondrial and Lipid Metabolic Gene NetworksDocket No. NEM-103-PCT Customer No. 51079

[0108] In parallel with detoxification responses, NL100P3 induced consistent upregulation of genes governing mitochondrial lipid utilization and energy metabolism, ech-6 (human ortholog: ECHS1) was upregulated, supporting enhanced mitochondrial P-oxidation and branched-chain amino acid catabolism. Lipid desaturation and long-chain polyunsaturated fatty acid metabolism were reflected by increased expression of fat-4 (human orthologs: FADS1 / 2 / 3).

[0109] Further reinforcement of fatty acid oxidation programs was observed through induction of acdh-3 (human ortholog: ACADSB) and acdh-1 (human ortholog: ACADS), which catalyze branched-chain, short-chain, and butyrate-associated fatty acid oxidation reactions. Mitochondrial respiratory capacity was additionally supported by upregulation of nuo-6 (human ortholog: NDUFB4), a component of mitochondrial complex I involved in oxidative phosphorylation. Together, these gene-level changes provide molecular support for enhanced mitochondrial energy utilization and metabolic efficiency.

[0110] Suppression of Nutrient-Sensing and Senescence-Associated Programs Supports Longevity-Associated Aging Hallmarks

[0111] In contrast to the induction of detoxification and metabolic genes, NL100P3 exposure resulted in coordinated downregulation of conserved genes associated with nutrient sensing, cellcycle regulation, and senescence-related signaling in Caenorhabditis elegans. Notably, rict-1 (C. elegans, human ortholog RICTOR), a core component of the mTORC2 complex that regulates anabolic signaling and cellular growth responses, was suppressed, consistent with attenuation of nutrient-responsive signaling. This observation is further supported by the absence of enriched mTOR-activating pathways and by pathway-level downregulation of PI3K–AKT–mTOR signaling.

[0112] Additional regulatory restraint was reflected by downregulation of cdk-9 which governs transcriptional elongation and cell cycle progression and lin 9 a component of the Additional regulatory restraint was reflected by downregulation of cdk-9 (CDK9), which governs transcriptional elongation and cell-cycle progression, and lin-9 (LIN9 a component of the DREAM complex involved in Gl / S checkpoint control. Suppression of lin-42 PERl / 2 / 3 which integrates circadian rhythm and cell-cycle regulation, further indicates modulation of temporal growth and stress-response coordination. Collectively, suppression of these conserved regulatory genes is consistent with attenuation of nutrient-sensing and pro-senescent signaling programs.Docket No. NEM-103-PCT Customer No. 51079

[0113] Within the framework of conserved aging biology, these transcriptional features map to a defined subset of longevity-associated aging hallmarks, including deregulated nutrient sensing and cellular senescence, as described by Lopez-Otin et al. (Cell, 2023). In parallel, genes annotated to chronic inflammation and epigenetic regulation exhibited coordinated modulation, consistent with engagement of antagonistic and integrative hallmarks that support cellular maintenance and stress adaptation. Visualization of gene-level log₂ fold changes grouped by hallmark category demonstrates directional trends within these domains, providing hallmark-level context for the metabolic and stress-resilient transcriptional state induced by NL100P3 in C. elegans.

[0114] Integrative Mo A

[0115] The transcriptional response to NL100P3 reflects a coordinated rebalancing of cellular energy handling and growth control rather than independent modulation of isolated pathways. At the metabolic level, induction of mitochondrial P-oxidation enzymes (ech-6, acdh-1, acdh-3) increases flux of fatty acids into acetyl-CoA, thereby feeding the TCA cycle and supporting oxidative phosphorylation (nuo-6). This shift toward lipid-derived energy production establishes a high-efficiency mitochondrial state that sustains ATP generation without engaging anabolic growth programs.

[0116] Concurrently, activation of NRF2-linked detoxification genes (gst-1, ugt-6, ugt-62, cyp-14A5) enhances redox buffering and xenobiotic clearance, reducing oxidative burden generated by increased mitochondrial activity. This detoxification module functionally stabilizes the metabolic state by limiting stress-induced signaling that would otherwise reactivate growth or damageresponse pathways.

[0117] Importantly, this metabolically active yet stress-buffered state is reinforced by suppression of nutrient-sensing and growth-promoting regulators. Downregulation of rict-l (RICTOR) constrains mTORC2 assembly, thereby dampening AKT-mediated nutrient and growth signaling. Reduced expression of translational regulators (eif-4E, eif-4A, eif-4G further limits mTORCl-dependent protein synthesis, effectively decoupling energy availability from cellular growth commitment.

[0118] This restraint is extended to higher-order regulatory control through suppression of cdk-9 and lin-9, which limits transcriptional elongation and Gl / S cell-cycle progression, and Un-42, which integrates circadian timing with growth responsiveness. Together, these regulatory nodesDocket No. NEM-103-PCT Customer No. 51079 enforce a cellular state that favors maintenance and metabolic efficiency over proliferation or hypertrophy.

[0119] Collectively, NL100P3 drives a self-reinforcing biological program in which enhanced mitochondrial lipid utilization and detoxification capacity are functionally coupled to suppression of nutrient-sensing, translational, and cell-cycle drivers. This coordinated regulation stabilizes a low-growth, high-efficiency metabolic state that is mechanistically aligned with longevity-associated biology in C. elegans, providing a coherent causal framework linking gene-level regulation to aging-relevant functional outcomes.

[0120] Examples

[0121] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0122] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C. or is at ambient temperature, and pressure is at or near atmospheric.

[0123] Example 1: Differential Gene Expression Study

[0124] Transcriptomic analysis of individual bioactives was conducted

[0125] Methods

[0126] RNA will be extracted from frozen worm samples using the Direct-zol RNA Miniprep kit (Zymo Research, cat# R2050). Two to three independent biological replicates will be prepared for each treatment. Library preparation and next-generation sequencing will be performed to generate strand-specific (second strand cDNA synthesis with dUTP) 100 bp paired-end reads. Cleaned reads, with adapter sequences, contamination, and low-quality reads removed via Trimmomatic (version 0.39) [1], will be obtained from BGI. These reads will be assessed for quality using FastQC (Babraham Bioinformatics) [2], ensuring no over-represented sequences or adapter sequences, and median per base quality scores consistently above 30. Transcript-level abundancesDocket No. NEM-103-PCT Customer No. 51079 will be estimated via pseudo-alignment to the C. elegans reference transcriptome (Ensembl release 108) [3] using Kallisto (version 0.48.0) [4], Gene counts will then be inferred using the HISAT2 R package (version 1.6.0) [5], and lowly expressed genes (those with counts less than 10 in every sample) will be filtered out for downstream analysis.

[0127] Differential gene expression analysis will be performed using edgeR (version 3.42.4) [6] in R (version 4.3.1). Principal component analysis (PCA) of the top 500 most variable genes, with variance stabilizing transformed counts used as input, will be conducted for unsupervised clustering of samples. Wald tests will then be used to test for differential gene expression. Pairwise comparisons will be made between no-treatment controls and treatment groups, as well as between samples from each individual ingredient. The Benjamini-Hochberg procedure will be used to adjust p-values to control the false discovery rate (FDR) [7], Significant gene expression changes will be defined at the adjusted p < 0.05 level. Functional characteristics of differentially expressed gene lists will be elucidated by performing over-representation analysis of Gene Ontology (GO) terms using the clusterProfiler R package (version 4.8.2) [8], Each GO sub-category (biological process, cellular component, molecular function) will be considered, with the corresponding background gene list consisting of genes input into differential expression testing. Enriched GO terms will be defined as those with a Benjamini-Hochberg corrected p < 0.05 [7], Network enrichment analysis will be performed using Cytoscape CyP3 R package (version 3.10.1) [9], Briefly, Cytoscape is an open-source platform for visualizing molecular interaction networks and integrating them with experimental data. Networks will be constructed by mapping gene expression values onto nodes, and functional relationships will be explored using built-in enrichment analysis tools and layout algorithms to identify patterns and clusters. To perform tissue enrichment analysis, first differentially expressed genes were mapped to human orthologs using OrthoList2

[0010] with one-to-many mapping. Following this, the TissueEnrich software (version 1.16.0)

[0011] is used to analyze human-converted gene expression data to identify tissue-specific genes based on RNA-seq expression profiles. Genes with transcripts per million (TPM) values greater than 1 will be classified as tissue-enriched, and enrichment analysis will be performed by comparing gene expression levels across multiple tissues to identify those with elevated or preferential expression in specific tissue types.

[0128] Fig. 1 is a table showing the Differential Gene Expression Summary. Human homologs were mapped to C. elegans genes using OrthoList with one-to-many mappings. Total DEGsDocket No. NEM-103-PCT Customer No. 51079 include all genes with adj. p value <0.1 and abs(Log2FoldChange) > 0.5. Small effects on gene expression may be due to time point of collection (not long enough).

[0129] Figs. 2A-2C are the gene expression overlap analysis of C. elegans. Fig. 2A is a graph showing the full data overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 2B is a graph showing the upregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 2C is a graph showing the downregulared genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A.

[0130] Figs. 3A-3C are the gene expression overlap analysis of H. sapiens. Fig. 3A is a graph showing the overlap of significant genes for Fisetin, Rosmarinic Acid, and Urolithin A. Fig. 3B is a graph showing the upregulated genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A. Fig.3C is a graph showing the downregulared genes overlap for Fisetin, Rosmarinic Acid, and Urolithin A.

[0131] Fig. 8A shows conceptual integrated mechanism-of-action model for NL100P3 under standard dietary conditions. The schematic synthesizes pathway- and gene-level transcriptional evidence into a unified biological framework linking enhanced mitochondrial lipid utilization and NRF2-associated detoxification with coordinated suppression of PI3K-AKT-mTOR nutrientsensing and growth signaling. Arrows indicate directionality of pathway engagement inferred from significantly regulated genes (|log2FC| > 0.5, adjusted / 9< 0.05).

[0132] This schematic presents an integrated mechanism by which NL100P3 coordinates energy metabolism, stress defense, and growth restraint into a single, self-reinforcing biological state. Transcriptional induction of mitochondrial P-oxidation and oxidative phosphorylation supports efficient ATP generation from lipid substrates, while concurrent activation of NRF2-linked detoxification and glucuronidation programs buffers oxidative and xenobiotic stress.

[0133] Critically, these metabolic and protective programs are coupled to suppression of PI3K-AKT-mTOR nutrient-sensing signaling, functionally decoupling energy availability from anabolic growth and translational commitment. This integration establishes a metabolically efficient, low-growth cellular configuration that prioritizes maintenance and stress resilience over proliferation, providing a mechanistic bridge between NL100P3 -induced transcriptional changes and longevity-associated biology.

[0134] Fig. 8B shows the Key Pathways: NL100P3 with a standard diet and shows that NL100P3 induces a highly organized transcriptional shift rather than isolated pathway changes. A strong activation of detoxification, redox, and mitochondrial lipid metabolism pathways was observed,Docket No. NEM-103-PCT Customer No. 51079 alongside coordinated suppression of nutrient- sen si ng, insulin, senescence, and mTOR-associated signaling. Together, this pattern indicates a transition toward a metabolically efficient, stress-resilient cellular state under standard dietary conditions.

[0135] The method of administering NL100P3 to Caenorhabditis elegans induces a coordinated transcriptional reprogramming characterized by upregulation of mitochondrial lipid β-oxidation, oxidative phosphorylation, PPAR-associated metabolic pathways, and NRF2-regulated detoxification programs, together with downregulation of nutrient-sensing and senescence-associated signaling pathways including PI3K-AKT-mTOR, thereby establishing a metabolic and stress-resilient cellular state associated with longevity-supportive biological processes in C. elegans.

[0136] Fig. 8C shows pathways, signals for claims, clinical endpoints: NL100P3 with a standard diet. This table organizes NL100P3 -responsive transcriptional pathways into a structured framework linking molecular regulation to claim-relevant biological signals and functional outcome domains. Pathways induced by NL100P3 converge on conserved detoxification and mitochondrial lipid metabolism programs, supporting enhanced xenobiotic handling and metabolic flexibility, while suppression of nutrient-sensing and senescence-associated pathways aligns with regulatory restraint of pro-aging signaling. Together, these coordinated transcriptional responses define a multi-axis biological profile supporting detoxification, metabolic health, and longevity-associated regulatory balance in C. elegans.

[0137] The transcriptional changes induced by NL100P3 in C. elegans exhibit functional convergence across detoxification, lipid metabolism, and nutrient-sensing pathways, resulting in coordinated enhancement of xenobiotic handling, mitochondrial energy utilization, and suppression of pro-senescent signaling programs.

[0138] The method of administering NL100P3 increases drug metabolism and NRF2 detoxification activation by increasing the activity of CYP450s (Cytochrome P450), UGTs (UDP-glucuronosyltransferase) for Liver detox, inflammation reduction. The method of administering NL100P3 increases biological oxidations and benefits phase I oxidation detox by increasing cytochrome P450 program and providing toxin clearance, antioxidant defense. The method of administering NL100P3 increases metabolism of xenobiotics by cytochrome P450 by benefiting xenobiotic clearance via detox enzymes by UGT / CYP induction and providing liver detox, metabolic reset. The method of administering NL100P3 increases glucuronidation to benefit PhaseDocket No. NEM-103-PCT Customer No. 51079 IT conjugative detoxification by increasing glucuronosyltransferase activation and providing hormonal detox, metabolic clearance. The method of administering NL100P3 increases NRF2 pathway to benefit antioxidant gene activation by NRF2-driven cytoprotective transcriptional program activation and providing enhanced oxidative resilience, inflammation suppression. The method of administering NL100P3 increases glutathione conjugation and benefits ROS detox via glutathione by enhancing glutathione-S-transferase-mediated clearance of reactive intermediates and providing redox balance, cellular protection. The method of administering NL100P3 increases glutathione transferase activity to benefit catalysis of GSH-conjugation by increasing GST enzymatic activity to provide detoxification, oxidative stress management.

[0139] The method of administering NL100P3 increases fatty acid metabolism / FAO and enhances mitochondrial β-oxidation by increasing FABP, carnitine enzymes to benefit energy metabolism, fat oxidation, metabolic flexibility. The method of administering NL100P3 increases PPAR signaling to benefit transcriptional control of lipid metabolism by the lipid utilization program; PPAR-driven energy repartitioning to provide metabolic remodeling, insulin sensitivity. The method of administering NL100P3 increases mitochondrial fatty acid beta-oxidation to benefit Mitochondrial energy efficiency increasing the induction of β-oxidation enzymes; mitochondrial lipid catabolism to provide anti-fatigue, mitochondrial health. The method of administering NL100P3 increases Electron transport / Oxidative phosphorylation to improve mitochondrial respiration by increasing OXPHOS gene programs to provide anti-aging, energy production to the subject. The method of administering NL100P3 decreases senescence-related programs to provide for Reduced pro-senescent signaling by decreasing p53 / CDKN axis to supports longevity, cellular rejuvenation of the subject. The method of administering NL100P3 decreases PI3K–AKT–mTOR signaling to provide suppressed anabolic / insulin signaling by mTOR, AKT, PI3K to provide Caloric restriction mimetic, anti-aging in the subject. Fig. 8D shows the genes affected by NL100P3 with a standard diet and provides gene-level evidence supporting the pathway-level transcriptional reprogramming induced by NL100P3. NL100P3 selectively activates conserved genes governing mitochondrial fatty acid oxidation, oxidative phosphorylation, and detoxification capacity, while concurrently suppressing genes linked to nutrient sensing, cell-cycle regulation, and senescence. The directional consistency across these gene classes establishes a molecular basis for enhanced metabolic efficiency, improved stress handling, and attenuation of pro-aging regulatory programs in C. elegans.Docket No. NEM-103-PCT Customer No. 51079

[0140] NL100P3 elicits directionally consistent regulation of conserved gene sets in C. elegans. including suppression of senescence-associated genes (cdk-9, lin-42, lin-9) and nutrient-sensing-associated genes (rict-4) and induction of genes governing mitochondrial lipid metabolism (fat-4, ech-6, acdh-1, acdh-3), oxidative phosphorylation (nuo-6), and phase I / II detoxification (gst-1, cyp-14A5, ugt-62, ugt-6, and cyp-13A5). The coordinated modulation of these gene classes provides molecular-level substantiation for the pathway-level transcriptional reprogramming observed with NL100P3 and their corresponding human homologs cdk-9 (CDK9), lin-42 (PER1 / 2 / 3), lin-9 (LIN9), rict-1 (RICTOR), fat-4 (FADS1 / 2 / 3), ech-6 (ECHS1), acdh-1 (ACADS), acdh-3 (ACADSB), nuo-6 (NDUFB4), ftn-2 (FTL, FTH1), gst-1 (GSTP1, GSTM1 / 2 / 3 / 4 / 5), cyp-14A5 (CYP2D6 / 7), ugt-62 (UGT2B10 / 28), ugt-6 (UGT3A1 / 2), and cyp-13A5 (CYP4B1 / A22).

[0141] Fig. 8E shows the Upregulated Markers with Benefit Indications of NL100P3 and highlights gene-level activation of conserved detoxification and mitochondrial metabolic programs in response to NL100P3. NL100P3 induces coordinated upregulation of phase I and phase II detoxification genes, including cytochrome P450 enzymes, glucuronosyltransferases, glutathione pathway components, and NRF2-associated targets, supporting enhanced xenobiotic clearance and redox homeostasis. In parallel, genes governing mitochondrial fatty acid β-oxidation, lipid desaturation, and oxidative phosphorylation are upregulated, indicating increased mitochondrial energy utilization and metabolic efficiency. Collectively, the directionally consistent activation of these gene classes provides functional evidence that NL100P3 engages established protective and metabolic pathways in C. elegans, reinforcing the pathway-level transcriptional response and supporting downstream health-relevant outcomes.

[0142] The method of administering NL100P3 upregulates the expression of cyp-14A5 (CYP2D6 / 7) to provide for detoxification by Phase I detox (oxidation of drugs & xenobiotics). The method of administering NL100P3 upregulates the expression ugt-6 (UGT3A1 / 2) to provide for detoxification by Phase II detox (glucuronidation, bile acid metabolism. The method of administering NL100P3 upregulates the expression cyp-13A5 (CYP4B1 / A22) to provide for detoxification by Lipid co-oxidation & xenobiotic metabolism. The method of administering NL100P3 upregulates the expression of ugt-62 (UGT2B 10 / 28) to provide for detoxification by Glucuronidation of amines (e.g., nicotine, drugs). The method of administering NL100P3 upregulates the expression of gst-1 (GSTP1, GSTM1 / 2 / 3 / 4 / 5) to provide for detoxification byDocket No. NEM-103-PCT Customer No. 51079 Redox balance (glutathione pathway). The method of administering NL100P3 upregulates the expression of ftn-2 (FTL, FTH1) to provide for detoxification by Iron homeostasis, (one of the NRF2 targets).

[0143] The method of administering NL100P3 upregulates the expression of ech-6 (ECHS1) to provide for metabolic health by Mitochondrial 0-oxidation & valine catabolism. The method of administering NL100P3 upregulates the expression of fat-4 (FADS1 / 2 / 3) to provide for metabolic health by Long-chain PUFA desaturation. The method of administering NL100P3 upregulates the expression of acdh-3 (ACADSB) for metabolic health by branched / short-chain fatty acid oxidation. The method of administering NL100P3 upregulates the expression of acdh-1 (AC ADS) for metabolic health by butyrate & short-chain FA oxidation. The method of administering NL100P3 upregulates the expression of nuo-6 (NDUFB4) for metabolic health by Mitochondrial Complex I (OXPHOS).

[0144] The method of administering NL100P3 downregulates the expression of cdk-9 (CDK9) for longevity by Cell cycle & transcription regulation. The method of administering NL100P3 downregulates the expression of Rict-1 (RICTOR) for longevity by guides the assembly and maintains the stability of the mTORC2 complex. The method of administering NL100P3 downregulates the expression of lin-9 (LIN9) for longevity by Gl / S checkpoint control (DREAM complex). The method of administering NL100P3 downregulates the expression of lin-42 (PER1 / 2 / 3) for longevity by Circadian rhythm & cell cycle interface.

[0145] Genes upregulated in response to NL100P3 correspond to conserved biological functions associated with detoxification capacity and mitochondrial energy metabolism. The directionally consistent activation of these gene classes indicates engagement of established protective and metabolic programs in C. elegans, providing functional context for the observed transcriptional response.

[0146] Fig. 8F shows the downregulated markers with benefit indications and NL100P3 induces coordinated suppression of conserved genes governing nutrient-sensing, cell-cycle progression, and senescence-associated regulation. Downregulation of rict-1 (RICTOR) supports reduced mTOR complex activity, while suppression of cdk-9, lin-9, and lin-42 indicates restraint of transcriptional elongation, Gl / S checkpoint control, and circadian-linked growth signaling. Collectively, this gene-level pattern is consistent with attenuation of pro-growth and pro-senescent pathways and supports a longevity-associated transcriptional state in C. elegans.Docket No. NEM-103-PCT Customer No. 51079

[0147] Genes downregulated in response to NL100P3 are associated with conserved nutrientsensing, cell-cycle, and senescence-related regulatory functions. The coordinated suppression of these gene classes is consistent with attenuation of nutrient-sensing and pro-senescent signaling, providing molecular support for a longevity-associated transcriptional state in C. elegans.

[0148] References

[0149] 1. Bolger, AM., Lohse, M., & Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114-2120. https: / / doi.org / 10.1093 / bioinformatics / btu170

[0150] 2 Andrews, S. (2010). FastQC: A quality control tool for high throughput sequence data. Available online at: https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc /

[0151] 3. Howe, K. L., Contreras-Moreira, B., De Silva, N., Maslen, G., Akanni, W., Allen, J., Alvarez-Jarreta, J., Barba, M., Becerra, A., Cummins, C., Davidson, C., Dodiya, K., El Houdaigui, B., Fonseca, N. A., Gall, A., Garcia Giron, C., Grego, T., Guijarro-Clarke, C., Kaikala, V., & many others. (2023). Ensembl Genomes 2023: access to diverse genomics data sets. Nucleic Acids Research, 51(D1), D633-D639. https: / / doi.org / 10.1093 / nar / gkac1037

[0152] 4. Bray, N. L., Pimentel, H., Melsted, P., & Pachter, L. (2016). Near-optimal probabilistic RNA-seq quantification. Nature Biotechnology, 34(5), 525-527. https: / / doi.org / 10.1038 / nbt.3519

[0153] 5. Kim, D., Paggi, J. M., Park, C., Bennett, C., & Salzberg, S. L. (2019). Graph-based genome alignment and genotyping with HISAT2 and HISAT -genotype. Nature Biotechnology, 37(8), 907-915. https: / / doi.org / 10.1038 / s41587-019-0201-4

[0154] 6. Robinson, M. D., McCarthy, D. J., & Smyth, G. K. (2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics, 26(1), 139-140. https: / / doi.org / 10.1093 / bioinformatics / btp616

[0155] 7. Benjamini, Y., & Hochberg, Y. (1995). Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society: Series B (Methodological), 57(1), 289-300. https: / / doi.org / 10.1111 / j.2517-6161.1995.tb02031.x

[0156] 8. Wu, T., Hu, E„ Xu, S„ Chen, M., Guo, P., Dai, Z„ Feng, T., Zhou, L„ Tang, W„ Zhan, L., Fu, X., Liu, S., Bo, X., & Yu, G. (2021). clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation, 2(3), 100141. https: / / doi.org / 10.1016 / j.xinn.2021.100141Docket No. NEM-103-PCT Customer No. 51079

[0157] 9. Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B., & Ideker, T. (2003). Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Research, 13(11), 2498-2504. https: / / doi.org / 10.1101 / gr.1239303

[0158] 10. Kim, W., Underwood, R. S., Greenwald, I., & Shaye, D. D. (2018). OrthoList 2: a new comparative genomic analysis of human and Caenorhabditis elegans genes. G3: Genes, Genomes, Genetics, 8(10), 3379-3387. https: / / doi.org / 10.1534 / g3.118.200457

[0159] 11. Jain, A., & Tuteja, G. (2019). TissueEnrich: tissue-specific gene enrichment analysis. Bioinformatics, 35(11), 1966-1967. https: / / doi.org / 10.1093 / bioinformatics / btz015

[0160] Example 2; Metabolic health studies

[0161] Equipment and Reagents:

[0162] Nematode Growth Medium (NGM) Buffer, NGM Agar, 20 mg / mL E. coli OP50 resuspended in NGM, S-Basal Buffer, Erioglaucine disodium salt. An Infinity System XI 00 as disclosed in PCT Application Serial No. PCT / US2024 / 033889, herein incorporated by reference. C. elegans strain: N2 (wild type) were tested for Longevity, Cognitive Health, Muscle Health, Gut Health, Metabolic Health, and Gut Health for the methods and compositions.

[0163] Longevity Assessment:

[0164] Methodology:

[0165] Wild-type N2 strain were age-synchronized and cultivated on standard nematode growth medium (NGM) agar plates seeded with live Escherichia coli OP50. The worms were maintained on the agar plates until reaching the L4 larval stage. Infinity Chips, specialized microfluidic devices, were sterilized and conditioned according to standard protocols before use. Approximately 50 L4-stage worms were introduced into each chip, with each experimental group consisting of multiple chips ensuring that each experimental group contained at least 100 organisms.

[0166] Once loaded into the Infinity Chips, the worms were transitioned to a diet consisting of 20 mg / mL live E. coli OP50 in liquid NGM for the initial 24 hours on-chip, allowing them to progress to adulthood. Starting on day 4, each chip was processed using the Infinity XI 00 system to rinse the media from the chips along with eggs and progeny. This process involved flushing S-Basal buffer through the chips via the inlet and outlet ports. The integrated camera system on the InfinityDocket No. NEM-103-PCT Customer No. 51079 XI 00 captured high-definition videos of the worms in the chips during this step, which were used for key downstream analyses.

[0167] From day 4 until all worms were dead, the worms administered the appropriate diet for their experimental group. The control group received only the bacterial diet and appropriate vehicle control. These diets comprised precise concentrations of selected ingredients mixed with 20 mg / mL E. coli OP50 in NGM.

[0168] All experiments were conducted at a controlled temperature of 20 °C ± 1 °C to ensure consistency across groups.

[0169] Data Analysis:

[0170] Viability was assessed by analyzing the high-resolution videos of each chip after washing and quantifying live worms using automated proprietary Al based image analysis software. Longevity was monitored from day 4 until death, with survivorship data systematically recorded for each experimental group. Survival analysis was performed using GraphPad Prism to generate Kaplan-Meier plots and conduct log-rank tests comparing groups. Key metrics, including median and maximum lifespans and survival rates at specific time points, were calculated to illustrate the impact of dietary variations on C. elegans longevity.

[0171] Figs.7A-7B are graphs showing that NL100P3 exhibits lifespan enhancement in wild type cohort. Fig. 7C is a table showing the placebo, Urolithin A and NL100P3 treatment. ** Urolithin A 50 pM = Urolithin 11.4pg / mL.

[0172] Figure 14A shows the NL100P3 Extends Lifespan in C. elegans and survival was assessed in C. elegans using standard lifespan assays, with percent survival plotted over time. Across two independent studies, NL100P3 -treated animals exhibited a consistent rightward shift in survival curves and an increase in median lifespan compared to placebo. Median lifespan was extended by approximately 8-9%, with statistically significant differences observed between treatment and control groups.

[0173] Table 1: Lifespan analysis revealed a consistent extension of median survival in NL100P3-treated condition compared to placebo across two independent studies.Experimental % Statistical No. of worms Median SurvivalGroup Change Significance1Placebo 210 24 daysNL100P3 154 26 days 8.33% **** (p <0.0001)Docket No. NEM-103-PCT Customer No. 51079Placebo 170 22 daysNL100P3 164 24 days 9.09% **** (p <0.0001)

[0174] Cognitive Health Assessment

[0175] Methodology

[0176] The cultivation and loading of Caenorhabditis elegans (N2 strain) into Infinity Chips were performed as described in the longevity study methodology. In addition to the standard feeding regimen, prior to each daily feeding, organisms were exposed to a fluid stimulus on the Infinity XI 00 within the chips to induce a touch-like sensation. The stimulus procedure involved:

[0177] Baseline Activity Recording: Capturing a 90-second high-resolution video of worm activity before stimulation.

[0178] Mechanical Stimulation: Introducing a 5-second pulse of liquid NGM at a flow rate of 7 ml / minute to provide a mechanical stimulus.

[0179] Post-Stimulus Activity Recording: Recording another 90-second video to assess reflexive responses.

[0180] Following the stimulus, worms were fed according to their designated experimental group diets. Cognitive health was evaluated based on the reflexive responses, with stimulated movement quantified from days 5 to 10, during which the mechanosensory nervous system declines.

[0181] Data Analysis: as disclosed in PCT Application No. PCT / US2025 / 013068, herein incorporated by references.

[0182] Figs.8A-8B showNL100P3 suppress stimulated neural activity in adults. ** Phosphatidyl serine 50 uM = Phosphatidyl serine 19.3 ug / mL. Enrolled worm-subjects are the same as the longevity study.

[0183] Muscle Health Assessment

[0184] Methodology:

[0185] The cultivation and loading of Caenorhabditis elegans (N2 strain) into Infinity Chips were performed as described in the longevity study methodology. In addition to the standard feeding regimen, prior to each daily feeding, organisms were exposed to a fluid stimulus using the Infinity XI 00 system within the chips to induce a touch-like sensation. The stimulus and video recording protocol, as described in the cognitive health study, was utilized to evaluate muscle function impairment during mid-to-late life.Docket No. NEM-103-PCT Customer No. 51079

[0186] The organismal response post-stimulus was used to measure the total distance moved and the mean speed of the worm population. These metrics were employed to assess muscle health, with data quantified from day 12 to day 20, the period during which muscle health decline is most prominent in worms.

[0187] Data Analysis:

[0188] Post stimulus crawling speed and distance travel is measuring every day from early adulthood to late stage of the animal (before decrepitude). The % gain is calculated in distance travelled and speed with reference to control. Neuromuscular health is reported as the cumulative gain of distance travel and speed across life.

[0189] Gut Health Assessment

[0190] Leaky Gut Evaluation Approaches:

[0191] Natural Aging-Induced Leaky Gut: Assessed on day 20 of the organism's life.

[0192] Poor Diet-Induced Leaky Gut: Evaluated on day 12 by supplementing the diet with 2% glucose.

[0193] Methodology:

[0194] The cultivation and loading of Caenorhabditis elegans (N2 strain) into Infinity chips were performed as described in the longevity study methodology. The dietary regimen and feeding cycle remained consistent with the longevity study to ensure uniformity across different health assessments.

[0195] To evaluate the leaky gut phenotype, the following procedure was implemented:

[0196] On the designated evaluation day (day 20 for natural aging and day 12 for poor diet-induced), organisms were fed media containing the blue colored non-absorbable food dye Erioglaucine disodium. After dye administration, worms were incubated for three hours to allow for potential dye leakage. Excess dye was flushed out of the Infinity Chips by introducing S-Basal buffer through the inlet and outlet ports. Subsequently, worms were immobilized using sodium azide to facilitate brightfield imaging. The brightfield images of the worms were analyzed to determine the presence of dye within the body cavity versus its restriction to the intestinal lumen.

[0197] Data Analysis: as disclosed in PCT Application Serial No. PCT / US2024 / 033889, herein incorporated by reference.

[0198] Gut Health Assessment:Docket No. NEM-103-PCT Customer No. 51079

[0199] Healthy Gut: Dye remains confined to the intestinal lumen, indicating intact gut barrier integrity.

[0200] Leaky Gut: Dye leaks into the body cavity, signifying gut dysfunction.

[0201] The percentage of organisms exhibiting healthy versus leaky gut was calculated for each experimental group. Each experimental group consisted of multiple chips to ensure that each group contained at least 50 organisms.

[0202] Fig. 13A shows NL100P3 enhances intestinal barrier integrity and ~7% improvement in age induced leaky gut phenotype. Intestinal barrier integrity was assessed in C. elegans using a dye-leakage assay, in which leakage of a fluorescent dye from the intestine into the body cavity indicates impaired intestinal barrier function. A composite gut health score was used to quantify the extent of leakage in individual animals. Compared to placebo, NL100P3 treatment resulted in higher gut health scores, indicating reduced age-associated intestinal leakage.

[0203] Figs. 13B-13C shows intestinal lumen distention is reduced by NL100P3 treatment. In C. elegans, the width of the intestinal lumen was measured at two positions along the intestine, one near the front of the intestine below the pharynx and one near the rear of the intestine near the anus. Increased lumen width reflects abnormal intestinal distention, which can arise from impaired handling of intestinal contents, altered fluid balance, or intestinal stress. Treatment with NL100P3 reduced intestinal lumen widening at both positions compared to placebo, indicating a reduction in intestinal distention along the length of the gut. Intestinal distention is a conserved physical feature of gastrointestinal dysfunction across species. In humans, excessive distention of the gut is associated with sensations of bloating and abdominal discomfort. Accordingly, reductions in intestinal lumen distention observed in C. elegans are consistent with biological mechanisms relevant to distention-related gastrointestinal symptoms in humans.

[0204] The method of administering NL100P3 to a subject further comprises improving gut health under metabolic stress conditions of the subject.

[0205] Metabolic Health Assessment

[0206] Methodology

[0207] The cultivation and loading of the wild-type N2 strain into Infinity chips were performed as described in the longevity study methodology. The dietary regimen and feeding cycle remained consistent with the longevity study, with the modification that the diet includes additional supplementation with 2% glucose to assess metabolic health.Docket No. NEM-103-PCT Customer No. 51079

[0208] Survivorship was quantified from day 4 until all the worms were dead to monitor survival trends over this period.

[0209] Each experimental group consisted of multiple chips to ensure that each group contained at least 100 organisms. The control group received only the bacterial diet with glucose supplementation. All experiments were conducted at a controlled temperature of 20 °C ± 1 °C to ensure consistency across groups.

[0210] Data Analysis:

[0211] Viability was assessed by analyzing the high-resolution videos of each chip after washing and quantifying live worms using automated proprietary Al based image analysis software. Longevity was monitored from day 4 until death, with survivorship data systematically recorded for each experimental group. Survival analysis was performed using GraphPad Prism to generate Kaplan-Meier plots and conduct log-rank tests comparing groups. Key metrics, including median and maximum lifespans and survival rates at specific time points, were calculated to illustrate the impact of dietary variations on C. elegans longevity.

[0212] Figs. 6A-6B, 6C show that NL100P3 extend lifespan under chronic metabolic stress. ** Semaglutide 2.4 uM = Semaglutide 9.87 ug / mL. 16 day survival was used and semaglutide increased by 63.3%, while NL100P3 increased lifespan by 78.2%.

[0213] Figs. 6D-6E show that the binary compounds extend lifespan under chronic metabolic stress Rosmarinic acid 100 uM + Urolithin A 100 uM, Fisetin 100 uM + Urolithin A 100 uM, and Fisetin 100 uM + Rosmarinic acid 100 uM.

[0214] Fig. 6F show the full summary of the compounds tested for the metabolic health study.

[0215] Fig. 6G is a graph showing the Percent survival on day 16. FIG. 6H is a graph showing the Percentage change in survival on day 16.

[0216] Fig. 11A shows the NL100P3 maintains metabolic health benefits across analytical and supplier-grade materials. Metabolic health effects of NL100P3 were evaluated using both analytical-grade compounds and supplier-grade ingredient materials. Mean and median survival under high-sugar conditions were increased relative to placebo across all material sources, demonstrating that the metabolic health benefits of NL100P3 are preserved when using commercially sourced ingredients.

[0217] Table 2A: Metabolic health benefits of Analytical and Supplier grade materialsDocket No. NEM-103-PCT Customer No. 51079Median s Percent change in Mean Percent change in mean # of subjectsurvival median survival survival survivalPlacebo 212 14 13.56NL100P3 119 16 14.3 14.89 9.8NL100P3141 16 14.3 15.59 15.0Supplier 1NL100P3116 18 28.6 15.29 12.8Supplier 2

[0218] Fig. 11B shows NL100P3 maintains metabolic health benefits across a dose range and ingredient sources. NL100P3 was evaluated at multiple dose levels using supplier-grade ingredient materials sourced from two independent suppliers. The formulations differed in the source and form of the rosemary-derived component, including different rosemary extracts (50% rosmarinic acid) or rosmarinic acid preparations. Survival under high-sugar diet conditions was measured as an indicator of metabolic stress resilience. Across both suppliers and across the tested dose range, NL100P3 consistently increased mean and median survival relative to placebo. These results demonstrate that the metabolic health effects of NL100P3 are maintained across different doses and are robust to variation in ingredient source and formulation.

[0219] Table 2B: Dose range and Supplier Metabolic Health benefitsPercent Percent # of change in change in Median survival Mean survival worms median mean survival survival Placebo 194 14 14.47NL100P3 (Supplier 1) 150 mg 185 18 28.6 16.23 12.2 NL100P3 (Supplier 1) 15 mg 221 16 14.3 15.52 7.3 NL100P3 (Supplier 1) 1.5 mg 205 16 14.3 16.06 11.0 NL100P3 (Supplier 2) 150 mg 143 18 28.6 16.21 12.0 NL100P3 (Supplier 2) 15 mg 142 16 14.3 15.24 5.3 NL100P3 (Supplier 2) 1.5 mg 188 18 28.6 15.83 9.4Docket No. NEM-103-PCT Customer No. 51079

[0220] Table 2C: Composition and Human Dosage EquivalentsNL100P3 combo HumanHuman equivalent Human equivalent human equivalent equivalentdose Urolithin A dose Rosmarinic acid dose Fisetin dose(mg) (mg) (mg)(mg)NL100P3 (Supplier150 50 50 501)NL100P3 (Supplier15 5 5 5 1)NL100P3 (Supplier1.5 0.5 0.5 0.51)

[0221] NL100P3 was evaluated at multiple dose levels using supplier-grade ingredient materials sourced from two independent suppliers. The formulations differed in the source and form of the rosemary-derived component, including different rosemary extracts (50% rosmarinic acid) or rosmarinic acid preparations. Survival under high-sugar diet conditions was measured as an indicator of metabolic stress resilience. Across both suppliers and across the tested dose range, NL100P3 consistently increased mean and median survival relative to placebo. These results demonstrate that the metabolic health effects of NL100P3 are maintained across different doses and are robust to variation in ingredient source and formulation.

[0222] Oxidative Stress Resilience Assessment

[0223] Methodology

[0224] The cultivation and loading of Caenorhabditis elegans (N2 strain) into Infinity Chips were performed as described in the longevity study methodology. Following loading, the worms were maintained under the same dietary regimen and feeding cycle to ensure consistency across studies.

[0225] To assess oxidative stress, the following procedures were implemented:

[0226] After loading into the Infinity Chips, worms were fed a bacterial diet supplemented with the appropriate ingredient for a duration of 2 days. This period allows for the incorporation of the supplement into the worms' system. On day 6, chronic oxidative stress was induced by adding 5 mM paraquat, a herbicide known to generate reactive oxygen species (ROS), to the diet. The supplementation with both the selected ingredient and 5 mM paraquat continued daily until day 14, maintaining a consistent exposure to oxidative stress throughout this period.Docket No. NEM-103-PCT Customer No. 51079

[0227] Data Analysis:

[0228] Viability was assessed by analyzing the high-resolution videos of each chip after washing and quantifying live worms using automated proprietary Al based image analysis software. Longevity was monitored from day 4 until death, with survivorship data systematically recorded for each experimental group. Survival analysis was performed using GraphPad Prism to generate Kaplan-Meier plots and conduct log-rank tests comparing groups. Key metrics, including median and maximum lifespans and survival rates at specific time points, were calculated to illustrate the impact of dietary variations on C. elegans longevity.

[0229] Figs.7A-7C show that NL100P3 confer strong oxidative stress resilience. ** Ascorbic acid 10 mM = Ascorbic acid 1.76 mg / mL = 440 mg of human dose. % changes are calculated based on day 12 data.

[0230] Figure 10A shows the oxidative stress resilience data

[0231] Table 3: oxidative stress resilience dataNo. ofExperimental enrolled Median % Median % Statistical Group wormSurvival change Survival change Significance1subjectsDMSO Control 191 10 Days 10.63 Days - NL100P3 162 14 Days 40% 12.42 Days 22.7% **** (p<0.0001) Urolithin A 185 12 Days 20% 11.16 Days 15.2% **** (p<0.0001) Rosmarinic acid 187 10 Days 0% 10.61 Days 4.8% **** (P<0.0001) Fi setin 172 12 Days 20% 12.14 Days 20.0% **** (p<0.0001) Urolithin A + Fisetin 156 14 Days 40% 11.74 Days 16.0% **** (p<0.0001) Urolithin A +197 12 Days 20% 12.14 Days **** (P<0.0001) Rosmarinic acid 20.0%Rosmarinic acid +182 14 Days 40% 12.65 Days **** (p<0.0001) Fisetin 25.0%

[0232] Figure 10B shows improved survival under oxidative stress conditions.

[0233] Table 4: Oxidative stress resilience was assessed using a paraquat-induced stress assay, in which exposure to paraquat generates elevated oxidative stress and reduces survival. MedianDocket No. NEM-103-PCT Customer No. 51079 survival under oxidative stress was measured for NL100P3, individual components, and selected combinations. NL100P3 and several component combinations increased median survival compared to control, indicating enhanced resilience to oxidative stress.No. ofExperimental enrolled Median % Median % Statistical Group wormSurvival change Survival change Significance1subjectsDMSO Control 191 10 Days 10.63 Days - NL100P3 162 14 Days 40% 12.42 Days 22.7% «« (p<0.0001) Urolithin A 185 12 Days 20% 11.16 Days 15.2% **** (p<0.0001) Rosmarinic acid 187 10 Days 0% 10.61 Days 4.8% **** (P<0.0001) Fisetin 172 12 Days 20% 12.14 Days 20.0% **** (p<0.0001) Urolithin A + Fisetin 156 14 Days 40% 11.74 Days 16.0% (p<0.0001) Urolithin A +197 12 Days 20% 12.14 Days 20.0% **** (P0.0001) Rosmarinic acidRosmarinic acid +182 14 Days 40% 12.65 Days 25.0% **** (pO.0001) Fisetin

[0234] Figure 10C shows the additive survival benefits under oxidative stress with NL100P3 Components. Oxidative stress resilience was evaluated using a paraquat-induced stress assay, in which exposure to paraquat reduces survival. Individual components of NL100P3 (Urolithin A, Fisetin, and Rosmarinic acid) were tested alone and in combination. While each individual component provided a measurable increase in mean survival compared to control, combinations of components in 2 / 3 binary combinations produced greater survival benefits than individual ingredients alone. These results demonstrate additive or enhanced effects of the component combinations under oxidative stress conditions. Metabolic stress resilience

[0235] Fig. 12A shows reduction of sugar-induced fat accumulation in C. elegans, where the total fat content was quantified using a fluorescent fat-staining assay under a high-sugar diet. NL100P3 treatment reduced lipid accumulation compared to placebo. In C. elegans, total fat content was assessed under a high-sugar diet using a fluorescent fat-staining assay, with fluorescence intensityDocket No. NEM-103-PCT Customer No. 51079 serving as a quantitative measure of lipid accumulation. Worms treated with NL100P3 showed a marked reduction in fluorescence intensity compared to placebo, indicating lower overall fat accumulation under high-sugar conditions. A high-sugar diet induces excess lipid accumulation in C. elegans, modeling diet-induced metabolic stress. The reduction in fat staining observed with NL100P3 indicates an attenuation of sugar-induced fat accumulation.

[0236] Excess lipid accumulation under high-sugar conditions reflects conserved metabolic processes associated with dysregulated energy storage. Accordingly, reductions in diet-induced fat accumulation observed in C. elegans are consistent with biological mechanisms relevant to weight management and metabolic health in humans.

[0237] Fig. 12B shows consistent reduction in fat accumulation using supplier-grade ingredients. Supplier-grade rosemary extract, fisetin, and urolithin A reduced fat accumulation under high-sugar conditions, consistent with effects observed using analytical-grade compounds. Fat accumulation under a high-sugar diet was quantified using a fluorescent fat-staining assay. A combination of supplier-grade rosemary extract, fisetin, and urolithin A significantly reduced fat staining compared to vehicle control. These results are consistent with reductions in fat accumulation observed using analytical-grade compounds, demonstrating preservation of efficacy with commercially relevant ingredient materials.

[0238] Summary

[0239] 1. Longevity: NL100P3 showed positive effects on lifespan suggesting long-term benefit.

[0240] 2. Cognitive Health: NL100P3 suppressed stimulated neural activity strongly suggesting a calming effect.

[0241] 3. Muscle Health: NL100P3 does not show decline in age-induced muscle function.

[0242] 4 Gut Health: NL100P3 did improve gut health, regardless of age or diet induced.

[0243] 5. Metabolic Health: NL100P3 extended lifespan under metabolic stress significantly.

[0244] 6. Stress Resilience: NL100P3 improved resilience to oxidative stress significantly.

[0245] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0246] While the invention has been described in connection with various embodiments, it will be understood that the invention is capable of further modifications. This application is intended to cover any variations, uses or adaptations of the invention following, in general, the principles ofDocket No. NEM-103-PCT Customer No. 51079 the invention, and including such departures from the present disclosure as, within the known and customary practice within the art to which the invention pertains.

Claims

Docket No. NEM-103-PCT Customer No. 51079 CLAIMSWhat is claimed is:

1. A method of treating a subject, comprising: administering an effective amount of Fisetin, an effective amount of Urolithin A, and an effective amount of Rosmarinic acid, and synergistically improving the health of the subject.

2. The method of Claim 1, further comprising improving metabolic health of the subject.

3. The method of Claim 2, wherein metabolic health is selected from the group consisting of fat reduction under metabolic stress, improved blood sugar control, improved weight management (BMI), and improved detoxification.

4. The method of Claim 1, further comprising improving gastrointestinal health of the subject.

5. The method of Claim 4, wherein gastrointestinal health comprises gut health or leaky gut.

6. The method of Claim 1, further comprising increasing lifespan and longevity of the subject.

7. The method of Claim 6, wherein increasing the lifespan and longevity further comprises increasing longevity under metabolic stress.

8. The method of Claim 1, further comprising improving gut health under age-induced stress conditions of the subject.

9. The method of Claim 1, further comprising improving gut health under metabolic stress conditions of the subject.

10. The method of Claim 1, further comprising improving cognitive health or suppressing neural activity for a calming effect.

11. The method of Claim 1, further comprising inducing a coordinated transcriptional reprogramming characterized by upregulation of mitochondrial lipid P-oxidation,Docket No. NEM-103-PCT Customer No. 51079 oxidative phosphorylation, PPAR-associated metabolic pathways, and NRF2-regulated detoxification programs, together with downregulation of nutrient-sensing and senescence-associated signaling pathways including PI3K AKT mTOR, thereby establishing a metabolic and stress-resilient cellular state associated with longevity- supportive biological processes.

12. The method of Claim 1, further comprising inducing transcriptional changes for functional convergence across detoxification, lipid metabolism, and nutrient-sensing pathways, resulting in coordinated enhancement of xenobiotic handling, mitochondrial energy utilization, and suppression of pro-senescent signaling programs.

13. The method of Claim 1, further comprising directionally consistent regulation of gene sets, including suppression of senescence- and nutrient-sensing-associated genes and induction of genes governing mitochondrial lipid metabolism, oxidative phosphorylation, and phase I / II detoxification.

14. The method of Claim 1, further comprising upregulating genes correspond to conserved biological functions associated with detoxification capacity and mitochondrial energy metabolism.

15. The method of Claim 1, further comprising downregulating genes associated with conserved nutrient-sensing, cell-cycle, and senescence-related regulatory functions.

16. The method of Claim 1, wherein the subject is a mammal.

17. The method of Claim 1, wherein the dosage of the effective amount of Fisetin, the effective amount of Urolithin A, and the effective amount of Rosmarinic acid is between about 1.5 mg / day and about 150 mg / day.

18. A composition comprising: an effective amount of Fisetin, an effective amount ofDocket No. NEM-103-PCT Customer No. 51079 Urolithin A, and an effective amount of Rosmarinic acid, wherein the composition is present in a synergistic ratio effective to improve survival under metabolic stress, oxidative stress, or aging-related stress.

19. The composition of claim 18, wherein Fisetin, Urolithin A, and Rosmarinic acid deliver metabolic health benefits through independent biochemical pathways when administered as single ingredients.

20. A composition as claimed in Claim 18, wherein the combinations significantly extend lifespan under conditions of metabolic stress.

21. A composition comprising: a combination of an effective amount Fisetin and an effective amount Urolithin A, a combination of an effective amount Urolithin A and an effective amount Rosmarinic acid, a combination of an effective amount Rosmarinic acid and an effective amount Fisetin, or a combination of an effective amount Fisetin, an effective amount Rosmarinic acid, and an effective amount Urolithin A, wherein the combination is provided in a synergistic ratio effective to improve survival under metabolic stress, oxidative stress, or aging-related stress.

22. A composition as claimed in Claim 21, wherein the combination of an effective amount Fisetin and an effective amount Urolithin A improves gut health and leaky gut, or improves survival under metabolic stress and fat reduction under metabolic stress.

23. A composition as claimed in Claim 21, wherein the combination of an effective amount Rosmarinic acid and an effective amount Urolithin A improves survival under metabolic stress, improves fat reduction under metabolic stress, improves blood sugar control, improves weight management, improves detoxification, improves gut health and leaky gut, improves gastrointestinal health, improves cognitive health, or enhances anti-oxidantDocket No. NEM-103-PCT Customer No. 51079 benefits.

24. A composition as claimed in Claim 21, wherein the combination of an effective amount Fisetin and an effective amount Rosmarinic acid improves survival under metabolic stress, improves fat reduction under metabolic stress, improves blood sugar control, improves weight management, improves detoxification, or enhances anti-oxidant benefits.

25. A composition as claimed in Claim 21, wherein combination of an effective amount Fisetin, an effective amount Rosmarinic acid, and an effective amount Urolithin A improves survival under metabolic stress, improves fat reduction under metabolic stress, improves blood sugar control, improves weight management, improves detoxification, improves gut health and leaky gut, improves gastrointestinal health, improves cognitive health, or enhances anti-oxidant benefits.

26. A method of treating a subject under a standard diet, comprising: administering an effective amount of Fisetin, an effective amount of Urolithin A, and an effective amount of Rosmarinic acid in a synergistic ratio effective to improve health of the subject.

27. The method of Claim 26, further comprising increasing drug metabolism and NRF2 detoxification activation by increasing the activity of CYP450s (Cytochrome P450), UGTs (UDP-glucuronosyltransferase) for Liver detox, inflammation reduction.

28. The method of Claim 26, further comprising increasing biological oxidations and benefits phase I oxidation detox by increasing cytochrome P450 program and providing toxin clearance and antioxidant defense.

29. The method of Claim 26, further comprising increasing metabolism of xenobiotics by cytochrome P450 by benefiting xenobiotic clearance via detox enzymes by UGT / CYPDocket No. NEM-103-PCT Customer No. 51079 induction and providing liver detox and metabolic reset.

30. The method of Claim 26, further comprising increasing glucuronidation to benefit Phase II conjugative detoxification by increasing glucuronosyltransferase activation and providing hormonal detox and metabolic clearance.

31. The method of Claim 26, further comprising increasing NRF2 pathway to benefit antioxidant gene activation by NRF2-driven cytoprotective transcriptional program activation and providing enhanced oxidative resilience, inflammation suppression.

32. The method of Claim 26, further comprising increasing glutathione conjugation and benefits ROS detox via glutathione by enhancing glutathione-S-transferase-mediated clearance of reactive intermediates and providing redox balance, cellular protection.

33. The method of Claim 26, further comprising increasing glutathione transferase activity to benefit catalysis of GSH-conjugation by increasing GST enzymatic activity to provide detoxification, oxidative stress management.

34. The method of Claim 26, further comprising increasing fatty acid metabolism / FAO and enhances mitochondrial P-oxidation by increasing FABP, carnitine enzymes to benefit energy metabolism, fat oxidation, metabolic flexibility.

35. The method of Claim 26, further comprising increasing PPAR signaling to benefit transcriptional control of lipid metabolism by the lipid utilization program; PPAR-driven energy repartitioning to provide metabolic remodeling, insulin sensitivity.

36. The method of Claim 26, further comprising increasing mitochondrial fatty acid betaoxidation to benefit mitochondrial energy efficiency increasing the induction of P- oxidation enzymes; mitochondrial lipid catabolism to provide anti-fatigue, mitochondrial health.Docket No. NEM-103-PCT Customer No. 51079 37. The method of Claim 26, further comprising increasing electron transport / Oxidative phosphorylation to improve mitochondrial respiration by increasing OXPHOS gene programs to provide anti-aging, energy production to the subject.

38. The method of Claim 26, further comprising decreasing senescence-related programs to provide for reduced pro-senescent signaling by decreasing p53 / CDKN axis to supports longevity, cellular rejuvenation of the subject.

39. The method of Claim 26, further comprising decreasing PI3K–AKT–mTOR signaling to provide suppressed anabolic / insulin signaling by mTOR, AKT, PI3K to provide Caloric restriction mimetic, anti-aging in the subject.

40. The method of Claim 26, further comprising upregulating the expression of cyp-14A5 (CYP2D6 / 7) to provide for detoxification by Phase I detox (oxidation of drugs & xenobiotics). The method of administering NL100P3 upregulates the expression ugt-6 (UGT3A1 / 2) to provide for detoxification by Phase II detox (glucuronidation, bile acid metabolism.

41. The method of Claim 26, further comprising upregulating the expression cyp-13A5 (CYP4B1 / A22) to provide for detoxification by Lipid co-oxidation & xenobiotic metabolism.

42. The method of Claim 26, further comprising upregulating the expression of ugt-62 (UGT2B 10 / 28) to provide for detoxification by Glucuronidation of amines (e.g., nicotine, drugs).

43. The method of Claim 26, further comprising upregulating the expression of gst-1 (GSTP1, GSTM1 / 2 / 3 / 4 / 5) to provide for detoxification by redox balance (glutathione pathway).

44. The method of Claim 26, further comprising upregulating the expression of ftn-2 (FTL,Docket No. NEM-103-PCT Customer No. 51079 FTH1) to provide for detoxification by Iron homeostasis, (one of the NRF2 targets).

45. The method of Claim 26, further comprising upregulating the expression of ech-6 (ECHS 1) to provide for metabolic health by Mitochondrial P-oxidation & valine catabolism.

46. The method of Claim 26, further comprising upregulating the expression of fat-4 (FADS 1 / 2 / 3) to provide for metabolic health by Long-chain PUFA desaturation.

47. The method of Claim 26, further comprising upregulating the expression of acdh-3 (ACADSB) for metabolic health by branched / short-chain fatty acid oxidation.

48. The method of Claim 26, further comprising upregulating the expression of acdh-1 (ACADS) for metabolic health by butyrate & short-chain FA oxidation.

49. The method of Claim 26, further comprising upregulating the expression of nuo-6 (NDUFB4) for metabolic health by Mitochondrial Complex I (OXPHOS).

50. The method of Claim 26, further comprising downregulating the expression of cdk-9 (CDK9) for longevity by Cell cycle & transcription regulation.

51. The method of Claim 26, further comprising downregulating the expression of Rict-1 (RICTOR) for longevity by guides the assembly and maintains the stability of the mTORC2 complex.

52. The method of Claim 26, further comprising downregulating the expression of lin-9 (LIN9) for longevity by Gl / S checkpoint control (DREAM complex). The method of administering NL100P3 downregulates the expression of lin-42 (PER1 / 2 / 3) for longevity by Circadian rhythm & cell cycle interface.

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