Compositions and methods for combating processes associated with inflammation and aging and supporting cellular energy and / or metabolism

Abstract

The contemplated compositions and methods are based on a combination of polyphenols commonly found in the Mediterranean diet, which reduces inflammatory signaling and aging-related gene expression, and supports mitochondrial biogenesis and cellular energy metabolism. Notably, the observed pleiotropic effects not only span multiple signaling pathways, but also show synergistic effects with respect to many markers associated with age-related energy decline, reduced immunity, and increased and sustained chronic subacute inflammation. From another perspective, the contemplated compositions represent a biochemically diverse molecular harmony that forms the basis for many of the benefits commonly observed in the Mediterranean diet.

Description

Related Applications

[0001] This application claims priority based on U.S. Provisional Patent Application Serial No. 63 / 215,716, filed on June 28, 2021, and U.S. Provisional Patent Application Serial No. 63 / 285,591, filed on December 3, 2021, and both applications are hereby incorporated by reference into this specification.

Technical Field

[0002] The field of the present invention is nutritional supplement compositions and methods, and in particular relates to polyphenols and polyphenol mixtures, and their use in reducing various age-related conditions such as mild / chronic inflammation, age-related decline in immunity, decline in energy metabolism, and / or obesity.

Background Art

[0003] The background description contains information useful for understanding the present disclosure. It is not admitted that any of the information provided herein is prior art, or is related to the presently claimed invention, or that any of the publications specifically or implicitly referred to are prior art.

[0004] All publications and patent applications in this specification are 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. If the definition or use of a term in an incorporated cited reference conflicts or contradicts the definition of that term as defined in this document, the definition of that term as defined in this document shall apply and the definition of that term in the cited reference shall not apply.

[0005] There are a fair number of types of vitamins and other isolated nutritional compounds, and the benefits of such compounds include immune support, anti-inflammatory effects, anti-aging effects, heart support, digestive support, and so on. Unfortunately, there is fairly little evidence to prove some of the aspects in which these benefits are claimed when taking vitamins and other isolated nutritional compounds. Similarly, when dietary supplements are extracts or powders of plant parts, various efficacy claims are made, but the actual efficacy is often unproven or not proven at all. Furthermore, isolated nutritional compounds and individual plant extracts or concentrates generally do not reflect a healthy diet.

[0006] In particular, there are specific geographical and ethnic dietary types related to overall health, longevity, and / or physical resilience, and such beneficial effects are well-documented and proven in fact. For example, the Mediterranean diet generally involves a reduction in cardiovascular risk factors (see, e.g., Nutrients 2018, 10, 379; doi:10.3390 / nu10030379), a reduction in inflammatory and metabolic biomarkers, a reduction in the risk of Alzheimer's disease (see, e.g., J Alzheimers Dis. 2010;22(2):483-492), and a decrease in certain inflammatory markers (see, e.g., Nutrients 2018, 10, 62; doi:10.3390 / nu10010062). One of the components commonly included in such diets is polyphenols, and various studies have been published on the specific effects of individual dietary polyphenols (see, e.g., Inhibitory Properties of Phenolic Compounds Against Enzymes Linked with Human Diseases: URL:dx.doi.org / 10.5772 / 66844) and selected colored polyphenols (see, e.g., Annu. Rev. Food Sci. Technol. 2020.11:10.1‐10.38). However, due to the large number and complexity of chemically different polyphenols, many studies have focused only on the specific biochemical effects of one type of polyphenol and its compounds, and have only provided general epidemiological information without clarifying the detailed molecular characteristics of the diet.

[0007] To supplement a diet with multiple polyphenols, various supplements are known. For example, Vital Reds (Gundry MD) markets a concentrated polyphenol powder blend from many red plant materials to increase energy and improve digestion. Such blends advantageously contain a variety of chemically different polyphenols. However, the selection of plant materials used as sources of polyphenols does not reflect intake from a typical diet. Similarly, as an antioxidant preparation for protecting cells from oxidative stress, Oxxynea from Fytexia, a commercial mixture of grape, olive, pomegranate, green tea, grapefruit, bilberry, and orange extracts, is provided (see, for example, Oxxynea from Fytexia). Although beneficial for reducing oxidative stress, the components that are the raw materials for such antioxidant preparations generally have not been shown to affect molecular signatures associated with (chronic subacute) inflammation, NFκB signaling, reduced energy metabolism, aging, age-related immune decline, and / or obesity.

[0008] Thus, despite the various nutritional supplements known in the art, all or almost all of them have various drawbacks. As a result, there is a need to provide improved compositions and methods for nutritional supplements, particularly those proven to counter (chronic subacute) inflammation, NFκB signaling, reduced energy metabolism, aging, age-related immune decline, and / or obesity. SUMMARY OF THE INVENTION

[0009] The present inventors have now found various compositions and methods that beneficially modulate various molecular signatures associated with age-related decline in immunity, inflammation, decreased energy metabolism, and / or obesity, for specific combinations of polyphenols commonly found in food items of the Mediterranean diet and / or materials rich in polyphenols (e.g., extracts or powders). As a result, the polyphenols and / or materials rich in polyphenols presented herein can be advantageously used in a nutrition-based approach to combat inflammaging, which may be characterized by one or more of age-related decline in immunity, increased or chronic inflammation, decreased energy metabolism, and / or obesity.

[0010] In one aspect of the subject matter of the present invention, the inventors contemplate a nutritional composition comprising a nutritionally acceptable carrier combined with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purple-blue colors, and in a particularly preferred embodiment, the red plant material, the green plant material, the orange-yellow plant material, and the purple-blue plant material are present in synergistic amounts with respect to reducing inflammatory cytokine release in human leukocytes.

[0011] For example, the red plant material consists of apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant material consists of olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant material consists of onion extract, ginger extract, grapefruit extract, and carrot powder, and / or the purple-blue plant material consists of grape extract, blueberry extract, blackcurrant powder, and elderberry powder. From another perspective, the colored plant materials may be selected based on or be a part of the Mediterranean diet.

[0012] In some embodiments, the inflammatory cytokine release is at least one, or at least two, or at least three of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and isoprostane release. In other embodiments, the inflammatory cytokine release is the release of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and isoprostane.

[0013] It should be further understood that the compositions presented herein can further reduce the expression of NFκB, increase glucose uptake into cells, increase mitochondrial biogenesis in cells, reduce oxidative damage by reactive oxygen species, reduce the expression of inflammatory adipokines, and / or reduce the expression of one or more aging-related genes.

[0014] Most commonly, the composition is formulated for oral administration and is formulated as a bulk material or in single-dose units, but this is not necessarily required. For example, the single-dose unit can contain 50-1000 mg of the composition and can be formulated as a capsule, gummy, or bulk powder. It can also be readily understood that the contemplated composition may further contain vitamins, trace elements or minerals in food, probiotics, and / or prebiotics. Similarly, the contemplated composition may also contain niacin, niacinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and / or a nutritionally acceptable CD38 inhibitor.

[0015] Accordingly, the contemplated composition is considered to be effective in treating or alleviating symptoms associated with inflammatory conditions, metabolic disorders, neurological conditions, cardiovascular conditions, aging, and / or oxidative stress.

[0016] Accordingly, the inventor contemplates a method of supporting the health of a subject to whom the composition shown herein is administered. For example, the composition may be administered in an amount effective to treat or alleviate symptoms associated with inflammatory conditions, metabolic disorders, neurological conditions, cardiovascular conditions, aging, and / or oxidative stress. Accordingly, the administration may be carried out over at least 30 days and / or at a daily dose of 50 to 1000 mg. Thus, the contemplated composition may be used to support the health and healthy aging of a subject by oral administration of the composition.

[0017] Accordingly, in a further aspect of the subject invention, the inventor also contemplates a method of reducing inflammaging in a subject, said method comprising administering to the subject a nutritionally acceptable carrier in combination with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purple-blue colors, wherein the combination of said plant materials reduces the release of at least one inflammatory cytokine in human cells and reduces the expression of at least one aging-related gene in human cells.

[0018] In certain embodiments, the red plant material consists of apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant material consists of olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant material consists of onion extract, ginger extract, grapefruit extract, and carrot powder, and / or the purple-blue plant material consists of grape extract, blueberry extract, blackcurrant powder, and elderberry powder. Thus, the red plant material, the green plant material, the orange-yellow plant material, and the purple-blue plant material may be part of a Mediterranean diet or selected based on the Mediterranean diet.

[0019] Most typically, the inflammatory cytokine is selected from the group consisting of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and isoprostane. In a particularly preferred embodiment, the red plant material, the green plant material, the orange-yellow plant material, and the purple-blue plant material are present in synergistic amounts with respect to reducing inflammatory cytokine release in human cells. With regard to the aging-related gene, it is contemplated that the aging-related gene is selected from the group consisting of HGF, c-fos, p16INK, and p21.

[0020] Advantageously, the reduction of inflammaging consists of the reduction of the age-related decline in immunity, the reduction of the age-related decline in energy metabolism, the reduction of the age-related decline in mitochondrial biogenesis, the reduction of at least one symptom related to inflammation, and / or the reduction of at least one symptom related to metabolic syndrome.

[0021] Furthermore, in at least some embodiments, it should be understood that administration of the composition further advantageously reduces the expression of NFκB, increases glucose uptake into cells, increases mitochondrial biogenesis within the cells, reduces oxidative damage by reactive oxygen species, reduces the expression of inflammatory adipokines, and / or increases intracellular ATP.

[0022] Administration to the subject consists of oral administration of a plurality of chemically different polyphenol-containing plant materials, and for example, a dosage between about 50 and 1,000 mg is preferred, but this is not necessarily required. Optionally, the plurality of chemically different polyphenol-containing plant materials may be formulated as tablets, drinks, or gummies. Further, the intended material may further contain vitamins, trace elements or minerals in food, probiotics, and / or prebiotics. Similarly, the combination of the plant materials may further contain niacin, niacinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and / or a nutritionally acceptable CD38 inhibitor.

[0023] In yet another aspect of the subject matter of the present invention, the inventors also contemplate a method of reducing inflammation in a subject comprising administering to the subject a nutritionally acceptable carrier in combination with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purple-blue colors. Preferably, the red plant material consists of apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant material consists of olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant material consists of onion extract, ginger extract, grapefruit extract, and carrot powder, and the purple-blue plant material consists of grape extract, blueberry extract, blackcurrant powder, and elderberry powder. In such a method, the combination of the plant materials, upon administration, reduces at least one inflammatory cytokine, reduces NFκF signaling, and / or reduces at least one inflammatory adipokine in the subject. The combination of the plant materials is preferably a synergistic combination with respect to the reduction of at least one cytokine, but this is not necessarily required.

[0024] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method for increasing glucose uptake in cells, comprising the step of contacting cells with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and violet-blue colors, wherein the red plant materials consist of apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant materials consist of olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant materials consist of onion extract, ginger extract, grapefruit extract, and carrot powder, and the violet-blue plant materials consist of grape extract, blueberry extract, stevia powder, and elderberry powder. In such a method, when a combination of the plant materials contacts the cells, glucose uptake into the cells increases. In particular, as detailed below, it has been observed that the increase in glucose uptake into the cells is equal to or greater than the increase in glucose uptake (under otherwise the same conditions) when using a thiazolidinedione agent such as rosiglitazone.

[0025] In a further aspect of the subject matter of the present invention, the inventors also contemplate a method for increasing the ATP level in cells, comprising the step of contacting cells with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and violet-blue colors, wherein the red plant materials consist of apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant materials consist of olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant materials consist of onion extract, ginger extract, grapefruit extract, and carrot powder, and the violet-blue plant materials consist of grape extract, blueberry extract, stevia powder, and elderberry powder. In such a method, when a combination of the plant materials contacts the cells, the ATP level in the cells (particularly muscle cells) increases.

[0026] Thus, from another perspective, the present inventors contemplate a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purple-blue colors, which are used to reduce at least one symptom of inflammatory aging in an individual who ingests the plant material.

[0027] Similarly, the present inventors also contemplate the use of the nutritional compositions presented herein to reduce inflammatory cytokine release in human leukocytes, reduce NFκB signaling, increase glucose uptake into cells, increase mitochondrial biogenesis in cells, reduce oxidative damage by reactive oxygen species, reduce the expression of inflammatory adipokines, and / or reduce the expression of at least one aging-related gene.

[0028] Various objects, features, aspects, and advantages of the subject matter of the present invention will become more apparent from the following detailed description of the preferred embodiments, together with the accompanying drawings in which like numerals represent like components.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0055] The inventor has found that a specific combination of polyphenol-containing substances (and the polyphenols contained therein) strongly alters numerous biomarkers associated with increased or chronic inflammation, NFκB signaling, decreased energy metabolism, aging, age-related decline in immunity, and / or obesity. In particular, such a combination closely matches the components included in the Mediterranean diet and polyphenol-containing substances (and polyphenols). In view of these findings, the inventor contemplates various compositions for use in dietary supplements and other nutritional products, compositions, and medicinal diets, and thus for use in medicine.

[0056] Based on their extensive research, the inventor has discovered that, as detailed below, by preparing a specific blend of selected plant materials (and the polyphenols contained therein) commonly found in the Mediterranean diet, it is possible to mimic the benefits of the Mediterranean diet, as demonstrated by changes in various biomarkers related to inflammatory aging and cellular energy. Among other biomarkers, in particular, various inflammatory cytokines and transcription factors (e.g., TNF-alpha, IL-6, PGE2, isoprostanes, NFκB), various adipokines (e.g., leptin, oncostatin M, resistin, FGF21, HGF, IL-11), mitochondrial biosynthesis, cellular glucose uptake, intracellular ATP, antioxidant capacity measured by reactive oxygen species, and biomarkers related to the expression of various aging-related genes are contemplated.

[0057] In particular, the inventor has found that the composition which is the subject of the present invention not only inhibits or reduces the production and secretion of inflammatory cytokines acting on different inflammatory pathways, but also down-regulates the transcription factors essential for the inflammatory response. More surprisingly, such regulatory effects on inflammatory components are particularly remarkable and synergistically enhanced when a plurality of chemically different polyphenol-containing plant materials of red, green, orange-yellow, and purple-blue colors are combined. Even more surprisingly, the inventor has found that the combination of chemically different polyphenol-containing plant materials brings about further beneficial effects far exceeding the anti-inflammatory effect. In particular, and as shown in more detail below, the composition presented herein has a remarkable effect of increasing glucose uptake into cells and mitochondrial biogenesis, increasing ATP levels while providing a significant antioxidant effect. Furthermore, this composition also showed a significant effect on specific adipokines related to inflammation and energy metabolism. Finally, such a composition also decreased the gene expression of various aging-related genes. These effects, especially when taken together, seem to suggest a beneficial molecular mechanism equivalent or similar to that commonly observed in the Mediterranean diet. Therefore, without wishing to be bound by any particular theory or hypothesis, the inventor believes that the composition presented herein represents the harmony underlying the biochemically diverse molecules that underlie many of the benefits seen in the Mediterranean diet. In such a situation, it should be understood that various other dietary patterns related to longevity and healthy aging (Blue Zone diet) can be a source of raw materials containing polyphenols that enhance the pathways and signaling required for health and lifespan in cooperation with other substances.

[0058] Preferably, as the blend, combinations of colored plant materials belonging to a plurality of different color groups (e.g., at least two, at least three, or at least four types) are contemplated, in particular plant materials that are red, green, orange / yellow, and / or purple / blue. For example, in one embodiment of such a composition, as detailed below, the polyphenol-containing product / extract is obtained from red raw materials including apple extract, pomegranate extract, tomato powder, beetroot, olive extract, rosemary extract, unroasted coffee bean extract, green raw materials such as kale, onion extract, ginger extract, grapefruit extract, carrot, etc., orange / yellow raw materials, and purple / blue coloring raw materials including grape extract, blueberry extract, bilberry, elderberry, and the specific components and ratios are detailed below. From another perspective, the contemplated composition contains a number of polyphenols belonging to at least two, at least three, or at least four different polyphenol classes, including organic acids, phenols, flavonols, flavanols, anthocyanins, chlorogenic acid, betacyanins, etc. It will be readily understood that the specific choice of plant material depends on the desired (polyphenol) components in the plant material and its effect on specific biological systems and / or signaling pathways.

[0059] Of course, it should also be understood that the plant material can be provided in various forms, including the whole or a part (e.g., roots, fruits, leaves, etc.) of the plant material in fresh or dried form, juice or extract from the plant material or a part thereof in fresh or dried form, and aqueous or aqueous / alcoholic extracts and chromatographic fractions of the aforementioned plant material. Furthermore, it should be noted that one or more polyphenols of the plant material may sometimes be provided as purified (naturally isolated or synthetic) chemical entities having a chemical purity of generally at least 90%, at least 95%, at least 98%, or at least 99%. However, in most embodiments, the plant material is a complex mixture that provides a combination of desired biological effects on numerous different molecular entities (e.g., enzymes, receptors, ion channels), and at least some of these effects are recognized as synergistic biological effects (e.g., at least one, at least two, or at least three, etc.). Additionally, the biological effects on specific molecular entities are also intended to be complementary and in some cases even synergistic in biological functions. Therefore, it should be understood that, based on the tests and desired targets described in more detail below, the compositions that are the subject of the present invention can be formulated to meet specific requirements. However, in a particularly preferred embodiment, compositions are contemplated that inhibit a plurality of targets (e.g., at least two, at least three, at least four, etc.) in a plurality of different (e.g., at least two, at least three, at least four, etc.) signaling pathways, preferably targets related to inflammation, immune response, and / or energy metabolism.

[0060] As a result, from another perspective, the mechanism of action of the contemplated composition is not limited to a single specific function (e.g., antioxidant), nor is it limited to a specific chemical category (e.g., vitamin). Instead, it is understood that the composition actually provides complementary and synergistic multiple biological effects across different metabolic and signaling pathways, and even other important cellular health functions such as mitochondrial biogenesis and glucose turnover. Thus, the contemplated compositions and methods target various biological systems including energy metabolism, immune function, inflammation, etc. From another perspective, the contemplated compositions and methods suppress inflammaging in the subjects who ingest the composition. As used herein, the term "inflammaging" refers to a complex collection of physiological events that includes at least one inflammatory component (e.g., inflammatory conditions, overexpression of inflammatory cytokines, and / or overactivation of inflammatory signaling pathways), and at least one typical age-related decline in cellular energy or mitochondrial biogenesis, and / or continuation or increase in the expression of aging-related genes. Furthermore, the plant material is also contemplated to provide various micronutrients to assist or complement the functions of polyphenols and other colored pigments in the composition.

[0061] Among other advantages, the compositions and methods presented herein provide significant complementary effects across multiple pathways that control inflammatory signaling, and as a result, will be understood to have a more desirable range of physiological effects. For example, as shown in more detail below, the intended compositions not only showed a significant decrease in inflammatory cytokine release, but also decreased NFκB signaling, PGE2, adipokines, and isoprostane release, which are additional inflammatory mediators that act on separate signaling pathways. Notably, sub-blends of the intended compositions showed some effect in modulating the above inflammatory mediators, but combining the sub-blends showed a strong synergistic effect in all of the above elements. Furthermore, the compositions contemplated herein further showed a significant antioxidant effect determined by a decrease in reactive oxidative species (ROS), thereby further reducing potential cellular stress and inflammatory signaling.

[0062] Similarly notable is that the intended compositions affected cellular energy management, and in particular had a significant impact on glucose uptake, mitochondrial biogenesis, and ATP levels. In fact, the intended compositions resulted in an improvement in glucose uptake that was equivalent to, and in some cases exceeded, the effects observed with rosiglitazone, a common anti-diabetic drug. Advantageously, such an increase in energy uptake did not lead to an increase in free radical production, but the intended compositions significantly stimulated mitochondrial biogenesis and increased intracellular ATP levels. Therefore, combining these treatments can be seen as counteracting the age-related decline in energy metabolism. Furthermore, with regard to age-related decline, the inventors have also found that the intended compositions decrease the expression of numerous aging-related genes.

[0063] As a result, it will be understood that the compositions contemplated herein can be advantageously used as single products to support various aspects of health and healthy aging, such as supporting proper immune function, reducing inflammation, and supporting glucose levels (particularly glucose uptake into cells). Viewed from another perspective, it will be understood that the contemplated compositions can be used to treat or reduce symptoms associated with inflammatory conditions, metabolic disorders, neurological conditions, cardiovascular conditions, aging, and / or oxidative stress. In such a context, when used with a physiological function or state, the term "support" is noted to mean preventing a decrease in one or more components or the action of components with respect to the physiological function or state, at least partially reversing a decrease in one or more components or the action of components with respect to the physiological function or state, maintaining the normal function of one or more components or the action of components with respect to the physiological function or state, preventing abnormal overaction (or overexpression) of one or more components with respect to the physiological function or state, and / or at least partially reversing abnormal overaction (or overexpression) of one or more components with respect to the physiological function or state. Alternatively, the compositions contemplated herein can also be combined with other dietary supplements and / or vitamins to provide beneficial effects not obtainable with the dietary supplement or vitamin alone.

[0064] Regarding the molecular mechanisms underlying the various effects observed using the contemplated compositions, the inventors investigated various potential targets that can be favorably regulated by the contemplated compounds, including various cytokines, NF-κB, various adipokines, glucose uptake, mitochondrial biogenesis, ATP levels, reactive oxygen species, expression of aging-related genes, and acetylcholinesterase.

[0065] Interleukin-6: Interleukin-6 (IL-6) is a multifunctional cytokine that controls immune responses, acute-phase reactions, and hematopoiesis and is thought to play a central role in host defense mechanisms. IL-6 is a pleiotropic cytokine produced by various cells. It acts on a wide range of tissues and exhibits growth promotion, growth inhibition, or differentiation depending on the nature of the target cells. In particular, IL-6 is not constitutively produced in normal cells but its expression is readily induced by various cytokines, bacterial components such as lipopolysaccharides, or viral infections. IL-6 is generally produced at the site of inflammation. IL-6 binds to its soluble receptor sIL-6Rα and determines the transition from acute to chronic inflammation by changing the nature of leukocyte infiltration (from polymorphonuclear neutrophils to monocytes / macrophages). Furthermore, IL-6 stimulates T cells and B cells and promotes chronic inflammatory reactions. Therefore, attempts to downregulate IL-6 and IL-6 signaling are considered useful for the treatment of various chronic inflammatory diseases such as rheumatoid arthritis. As shown in more detail below, the contemplated compositions have a significant effect on IL-6 release and as a result are thought to contribute to the reduction or prevention of one or more symptoms commonly seen in (chronic and / or subacute) inflammatory diseases.

[0066] Tumor necrosis factor alpha (TNF-alpha): Tumor necrosis factor-alpha (TNF-alpha) is mainly produced by activated macrophages, T lymphocytes, and natural killer (NK) cells and is a central regulator of inflammation. TNF-alpha antagonists and inhibitors are an interesting approach in the treatment of inflammatory diseases in which TNF-alpha plays an important etiological role. Inhibition of TNF has proven to be an effective treatment for patients with rheumatoid arthritis and inflammatory diseases such as psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease. Furthermore, a preventive effect on septic shock and AIDS has also been suggested. Currently successful biological therapies include etanercept, infliximab, fully human monoclonal antibodies, adalimumab, and the like. However, because of the high cost and side effects of currently known synthetic antibody therapies, natural TNF inhibitors such as the compositions described herein are a means of attracting attention. In fact, as detailed below, the contemplated compositions have a substantial inhibitory effect on the secretion of TNF-alpha and are therefore thought to contribute to the reduction or prevention of one or more symptoms commonly seen in various (chronic and / or subacute) inflammatory diseases.

[0067] Prostaglandin E2 (PGE2): Prostaglandins (PGs) are major lipid mediators in animals and are synthesized from arachidonic acid with cyclooxygenase (COX-1 or COX-2) as the rate-limiting enzyme in vivo. Prostaglandin E2 (PGE2) is the most abundantly detected PG in various tissues and exerts various physiological and pathological effects through four receptor subtypes (EP1-4). Non-steroidal anti-inflammatory drugs such as aspirin and indomethacin exert a potent anti-inflammatory effect by inhibiting COX activity and consequently suppressing PGs. PGE2 controls acute and chronic inflammation, as well as autoimmune diseases, at the molecular and cellular levels. One of the major roles of PGE2 in acute inflammation, fever, and pain was understood to be vasodilation of vascular smooth muscle cells. Additional data later showed that PGE2 induces the activation of mast cells, resulting in enhanced vascular permeability and contributing to acute inflammation caused by PGE2. Furthermore, PGE2 promotes the differentiation of Th1 cells, the proliferation of Th17 cells, and the production of IL-22 from Th22 cells, exacerbating chronic inflammation and various autoimmune diseases. Additionally, PGE2 causes acute inflammation through the activation of mast cells via the EP3 receptor. PGE2 also induces chronic inflammation and various autoimmune diseases via the differentiation of T helper 1 (Th1) cells, the proliferation of Th17 cells, and the production of IL-22 from Th22 cells through the EP2 and EP4 receptors. In particular, as shown in more detail below, the inventors have discovered that the compositions presented herein have a significant inhibitory effect on the synthesis and release of PGE2. Therefore, the contemplated compositions not only contribute to the reduction or prevention of one or more symptoms commonly seen in various (chronic and / or subacute) inflammatory and autoimmune diseases, but are also thought to downregulate the exacerbated immune response.

[0068] Isoprostanes: Isoprostanes are mediators of the inflammation-promoting process that do not depend on COX-2 and enhance the perception of pain. Isoprostanes may further contribute to inflammation by increasing neutrophil adhesion to human venous endothelial cells and increasing endothelial cell permeability in response to oxidative stress. Therefore, the intended composition is considered to not only contribute to the reduction or prevention of one or more symptoms commonly seen in various (chronic and / or subacute) inflammatory diseases, particularly those related to oxidative stress, but also to contribute to the reduction or prevention.

[0069] Therefore, it will be understood that the compositions presented herein exhibit unexpected pleiotropic effects across multiple different inflammatory signaling elements and signaling pathways and can broadly and systemically reduce various (chronic and / or subacute) inflammatory conditions. Even more surprisingly, the inventors have found that the inhibition of IL-6, TNF-alpha, and PGE2 is based on the synergistic effects of the various sub-blends present in the compositions presented herein (see the following data), and that the inhibitory effects of the sub-blends alone are not substantially significant.

[0070] In addition to the effects on individual cytokines as described above, the inventors have also found that the compositions shown herein have a significant downregulatory effect on NF-κB. In such a situation, it will be understood that NF-κB induces the expression of various inflammatory genes including genes encoding cytokines, chemokines, and COX-2 and is also involved in the control of the inflammasome. Furthermore, NF-κB plays an important role in controlling the survival, activation, and differentiation of innate immune cells and inflammatory T cells and affects the innate immune response and the target-specific immune response.

[0071] NF-κB is highly activated at the inflammatory sites of various diseases and also induces the transcription of adhesion molecules, MMP, and inducible nitric oxide (iNOS). For example, in rheumatoid arthritis, NF-κB is overexpressed in the inflamed synovium, and its action is thought to promote the recruitment of inflammatory cells and the production of inflammatory mediators such as IL-1, IL-6, IL-8, and TNF-α. Both p50 and p65 are locally concentrated in the nuclei of synovial surface cells and mononuclear cells in the sublining area. Helicobacter pylori-associated gastritis is also marked by enhanced NF-κB activity in gastric epithelial cells, and the number of NF-κB-positive cells correlates with the degree of gastritis. Similarly, there is evidence of NF-κB activation in inflammatory bowel disease, in which case the lamina propria macrophages show activated p50, c-Rel, and especially p65. Inflammation associated with neurological diseases and atherosclerosis is also, in part, mediated by NF-κB. Furthermore, human inflammatory airway diseases are also associated with the expression of cytokines and adhesion molecules. This correlates with the activation of NF-κB in bronchial biopsies from asthmatic patients. In particular, an increase in NF-κB activity with nuclear localization was observed in airway epithelial cells rich in inflammatory cytokines, chemokines, iNOS, and Cox-2. Other NF-κB-related diseases include atherosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, and systemic inflammatory response syndrome. Therefore, a composition capable of regulating the expression of NF-κB may have important potential in addressing at least some of the symptoms associated with the inflammatory conditions described above. Therefore, again, the composition contemplated is thought to also contribute to the reduction or prevention of one or more symptoms commonly seen in various (chronic and / or subacute) inflammatory diseases.

[0072] Furthermore, particularly when an individual has an increased body mass index (BMI), or is overweight or obese, the composition contemplated may have a positive effect on adipokine signaling, as discussed and shown in more detail below.

[0073] Due to the global obesity epidemic, research aimed at understanding the biology of adipocytes, adipose tissue, and the phenomena occurring in the bodies of obese patients has received significant attention. Interestingly, there is a lot of evidence indicating that obesity causes chronic low-grade (subacute) inflammation, which may contribute to systemic metabolic dysfunction related to obesity-related diseases. In fact, adipose tissue functions as an important endocrine organ by secreting multiple bioactive substances known as adipose-derived secretory factors or adipokines, which may have pro-inflammatory or anti-inflammatory activities in some cases. Abnormal regulation of the production and secretion of these adipokines due to adipose tissue dysfunction may contribute to the development of obesity-related complications.

[0074] Inflammatory adipokines: In the obese state, the production of most adipokines is enhanced, and these inflammatory proteins usually function to promote metabolic diseases related to obesity. In addition to leptin, TNF-alpha, and IL-6, recently identified adipokines that promote inflammation include resistin, retinol-binding protein 4 (RbP4), lipocalin 2, IL-18, angiopoietin-like protein 2 (ANGPTL2), CC-chemokine ligand 2 (CCL2), CXC-chemokine ligand 5 (CXCL5), nicotinamide phosphoribosyltransferase (NAMPT), and others. Upregulation of these factors (and other factors) causes the development of a chronic inflammatory state and contributes to the occurrence of metabolic dysfunction.

[0075] Anti-inflammatory adipokines: In addition to the numerous inflammatory adipokines mentioned above, adipose tissue also secretes a few anti-inflammatory factors such as adiponectin, which has been intensively studied, and sFRP5, which was recently identified as an adipokine.

[0076] The present inventors have now found that, with respect to the secretion of adipokines, the compositions presented herein completely inhibit the secretion of IGFBP-1, leptin, oncostatin M, and resistin in TNF-alpha-induced adipocytes. The importance of these adipokines will be described later.

[0077] Leptin: Leptin, an adipokine, was identified in ob / ob mice by positional cloning of the obesity gene (ob, also known as Lep). Leptin controls feeding behavior through the central nervous system. Mice lacking leptin (ob / ob mice) exhibit hyperphagia (abnormal increase in food intake), obesity, and insulin resistance, but administration of leptin to ob / ob mice reverses these changes. Also, administration of leptin to lipoatrophic mice (with low leptin concentration due to lack of subcutaneous adipose tissue) improves metabolic abnormalities such as insulin resistance and hyperlipidemia. Leptin has also been shown to be effective in improving metabolic dysfunction in patients with lipodystrophy or congenital leptin deficiency. However, blood leptin concentration is positively correlated with fat mass, indicating the occurrence of leptin resistance, and obese individuals (persons) have high leptin concentration without the expected anorectic response. Leptin is structurally similar to the helical cytokine family including IL-2 and growth hormone 1 and is thought to have a pro-inflammatory effect. Indeed, leptin increases the production of TNF and IL-6 by monocytes and stimulates the production of CC-chemokine ligand (i.e., CCL3, CCL4, CCL5) by macrophages by activating the JAK2 (Janus kinase 2)-sTAT3 (signal transducer and activator of transcription 3) pathway. In monocytes as well, leptin stimulates the production of reactive oxygen species and promotes cell proliferation and migration responses. Leptin concentrations in serum and adipose tissue increase in response to inflammatory stimuli such as TNF and lipopolysaccharide (LP). Furthermore, leptin increases the production of TH1-type cytokines IL-2 and IFNγ by T cells and monocytes and suppresses the production of TH2-type cytokine IL-4, thus polarizing T cells toward the TH1 cell phenotype. Consistent with the above findings, leptin deficiency prevents liver injury in a hepatitis model mediated by T cells. Thus, leptin is generally recognized to act as an inflammatory adipokine.

[0078] Oncostatin M (OSM): OSM is a gp130 cytokine that heterodimerizes with gp130 and has a unique specific receptor, OSMR, which mediates most of the actions of OSM. OSM has substantial sequence identity with leukemia inhibitory factor (LIF) and can regulate various biological processes such as liver development and regeneration, hepatic insulin resistance and fatty liver, inflammation, dedifferentiation and remodeling of cardiomyocytes. Furthermore, OSM may contribute to the inflammatory state during obesity and be involved in the development of insulin resistance.

[0079] Resistin: Resistin is a member of the cysteine-rich resistin-like molecule (RELm) family and is associated with the activation of the inflammatory process. Resistin has been shown to induce insulin resistance in mice, and mice lacking resistin have low glucose production in the liver and thus low blood glucose levels after fasting. Resistin deficiency in ob / ob mice leads to increased obesity, but in these severely obese mice, glucose tolerance and insulin sensitivity are improved. The ability of resistin to regulate glucose metabolism is related to the activation of suppressor of cytokine signaling 3 (SOCS3), an inhibitor of insulin signaling in adipocytes. In animal model studies, it has consistently been shown that resistin promotes insulin resistance, but the evidence for this effect in humans is less clear. Resistin exists in two quaternary forms. An abundant high molecular weight hexamer and a less abundant but highly bioactive trimer that strongly induces hepatic insulin resistance. In human monocytes, transcription of the resistin gene (RETN) is induced by inflammatory cytokines such as IL-1, IL-6, and TNF, and in white adipose tissue, it is suppressed by rosiglitazone, a PPARγ agonist, suggesting that part of the anti-inflammatory action of rosiglitazone is mediated by attenuation of RETN transcription. More recently, studies of mice lacking endogenous resistin expression in adipocytes but expressing the human RETN transgene in macrophages have shown that macrophage-derived resistin contributes to insulin resistance in vivo through its pro-inflammatory properties. The pro-inflammatory properties of resistin in human monocytes are evident as resistin promotes the expression of TNF and IL-6 by these cells. Furthermore, resistin directly antagonizes the anti-inflammatory action of adiponectin in vascular endothelial cells by promoting the expression of the inflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM1), intercellular adhesion molecule 1 (ICAM1), and pentraxin 3, and promotes leukocyte adhesion.

[0080] IGFBP-1: Insulin-like growth factor-binding protein 1 (IBP-1), also known as placental protein 12 (PP12), is a protein encoded by the IGFBP1 gene in humans. This protein binds to both insulin-like growth factor (IGF) I and II and circulates in the plasma. When this protein binds, the half-life of IGF is extended and its interaction with cell surface receptors changes. The likelihood that the IGF system is involved in the development of cardiovascular diseases is increasing. The effects of circulating IGF on the vasculature are largely regulated by IGFBP, which controls access to cell surface IGF receptors. Since IGFBP-1 is subject to metabolic regulation by glucoregulatory hormones, it has been proposed as an acute regulator of IGF bioavailability. Post-translational phosphorylation of IGFBP-1 represents an additional mechanism for controlling IGF bioavailability, as it significantly increases the affinity for IGF-I.

[0081] In addition, the inventors have also found that, with respect to adipokine secretion, the compositions presented herein stimulate the secretion of ANGPT-L3, C-reactive protein, endocan, FGF-21, HGF, IGFBP-2, IL-11, RBP4, and pentraxin 2 in TNF-alpha-induced adipocytes (no observable stimulation in adipocytes induced by TNF-alpha alone), and the significance of these induced adipokines is described below.

[0082] RBP4: Serum RbP4 is a factor secreted from hepatocytes and is responsible for the systemic transport of retinol (vitamin A). Recently, it has been found that RbP4 is also secreted from adipocytes and macrophages. The expression of RbP4 is inversely correlated with the expression of glucose transporter type 4 (GLuT4, also known as sLC2A4), and administration of recombinant RbP4 to normal mice reduces insulin sensitivity. RbP4 is released from adipocytes and inhibits the phosphorylation of insulin receptor substrate 1 (IRs1) induced by insulin in an autocrine or paracrine manner. These data suggest that RbP4 is an important adipose tissue-secreted factor in the regulation of glucose homeostasis in type 2 diabetes models.

[0083] ANGPTL3: Angiopoietin-like protein 3 (ANGPTL3) is best known for its function as an inhibitor of lipoprotein and endothelial lipase. Genetic or pharmacological inhibition of ANGPTL3 has the ability to significantly reduce circulating lipoproteins, and since the cardioprotective effect of such inhibition has been demonstrated, ANGPTL3 has become a new therapeutic target for which antibody and antisense oligonucleotide (ASO) therapeutics are being clinically tested. This antibody is relatively selective for circulating ANGPTL3, but ASO also reduces intracellular proteins in hepatocytes, and evidence has emerged indicating the cell-autonomous function of ANGPTL3 in the liver. This includes the regulation of glucose and fatty acid uptake in hepatocytes, insulin sensitivity, LDL / VLDL remnant uptake, VLDL assembly / secretion, polyunsaturated fatty acid (PUFA) and PUFA-derived lipid mediator content, and gene expression.

[0084] Fibroblast Growth Factor 21: Fibroblast growth factor 21 (FGF21) is a protein encoded by the FGF21 gene in mammals. The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family, specifically a member of the endocrine subfamily that includes FGF23 and FGF15 / 19. FGF21 is the major endogenous agonist of the FGF21 receptor, which consists of the co-receptor FGF receptor 1 and β-Klotho. FGF21 is a hepatokine, that is, a hormone secreted from the liver, and regulates simple sugar intake and preference for sweet foods through signal transduction via FGF21 receptors in the paraventricular nucleus of the hypothalamus, and correlates with a decrease in dopamine neurotransmission in the nucleus accumbens. FGF21 stimulates glucose uptake in adipocytes but not in other cell types. This effect is additive with the activity of insulin. Treatment of adipocytes with FGF21 is associated with phosphorylation of FRS2, a protein that links the FGF receptor and the Ras / MAP kinase pathway. Injection of FGF21 into ob / ob mice increases Glut1 in adipose tissue. FGF21 also protects mice from diet-induced obesity when overexpressed in transgenic mice and lowers blood glucose and triglyceride levels when administered to diabetic rats. Administration of FGF21 to mice increases energy consumption, fat utilization, and lipid excretion.

[0085] Interleukin 11: Interleukin 11 (IL-11) is an anti-inflammatory cytokine, and receptors are present on most cell types and tissues throughout the body. Its anti-inflammatory properties are mediated through the suppression of cytokine synthesis, most of which is by preventing the activation of NF-κB. Adipose tissue synthesizes and secretes cytokines related to the establishment of insulin resistance, and since IL-11 has the ability to suppress cytokine synthesis, studies were initiated to clarify the signal transduction pathway initiated by IL-11 in adipose tissue.

[0086] Hepatocyte growth factor (HGF): Obesity and the accompanying chronic inflammation of adipose tissue cause insulin resistance associated with several pathological conditions such as hypertension and atherosclerosis. The body weight of wild-type mice fed a high-fat diet (HFD) for 14 weeks significantly increased with insulin resistance, but in HGF gene-transferred mice, body weight gain and insulin resistance were suppressed. In HGF gene-transferred mice, the accumulation of macrophages and the increase in inflammatory mediator levels in adipose tissue were significantly suppressed compared to wild-type mice. In wild-type mice administered with an HGF neutralizing antibody, obesity induced by HFD deteriorated in the glucose tolerance test. These gain-of-function and loss-of-function studies indicate that the increase in HGF levels induced by HFD plays a protective role against obesity and insulin resistance.

[0087] IGFBP-2: The proliferation of adipocyte precursors and the differentiation into mature adipocytes contribute to the development of obesity in mammals. IGF-I is a potent mitogenic factor and an important stimulator for adipocyte differentiation. The biological actions of IGF are tightly regulated mainly by the IGF-binding protein family (IGFBP) that exerts inhibitory effects. IGFBP-2 is the major binding protein secreted from differentiating white preadipocytes, suggesting its potential involvement in the development of obesity. Studies using transgenic mice overexpressing human IGFBP-2 under the control of its native promoter gene have shown that overexpression of IGFBP-2 is associated with a decrease in susceptibility to obesity and an improvement in insulin sensitivity. In littermate wild-type mice, glucose intolerance and increased blood pressure were induced with aging, but mice overexpressing IGFBP-2 were protected. Furthermore, when fed a high-fat and high-energy diet, mice overexpressing IGFBP-2 showed resistance to the development of obesity and insulin resistance. This lean phenotype was associated with a decrease in leptin concentration, an increase in glucose sensitivity, and a decrease in blood pressure compared to wild-type animals fed the same amount of high-fat diet. These findings suggest an important role of IGFBP-2 in obesity prevention.

[0088] Glucose uptake and mitochondrial biogenesis: In the elderly, protein synthesis in skeletal muscle can be resistant to the anabolic action of insulin. Insulin resistance is also associated with activation of the muscle proteolytic pathway and may lead to muscle loss. Furthermore, muscle is a major site of insulin-dependent glucose uptake, and a decrease in the surface area of muscle that takes up glucose via insulin can further exacerbate peripheral insulin resistance and lead to a vicious cycle. Oral insulin sensitizers have been reported to maintain muscle mass, but the same association with muscle strength has not yet been investigated. Interestingly, in type 2 diabetes, mitochondrial function in skeletal muscle is reduced and may be improved by peripheral insulin sensitizers. Against this background, the inventors have found that the compositions presented herein have a highly beneficial effect on glucose uptake in muscle cells, as detailed below. Furthermore, the compositions presented herein have the advantage of also improving mitochondrial function, particularly increasing mitochondrial biogenesis, as shown in more detail below. Thus, it should be appreciated that the compositions presented herein not only alleviate but rather reverse the age-related decrease in glucose uptake into muscle and even increase mitochondrial biogenesis.

[0089] To determine whether the compositions presented herein can increase the intracellular energy load, tests were also conducted on intracellular ATP levels. Indeed, as shown in detail below, the levels of intracellular ATP were improved, particularly when the concentration or dosage of the composition of interest was high.

[0090] Reactive oxygen species: Generally, reactive oxygen species (ROS) include derivatives of oxygen molecules as by-products normally generated in aerobic metabolism. When the formation of different ROS increases, it generally leads to molecular damage ("oxidative stress"). Hydrogen peroxide (H2O2) and superoxide anion radical (O2-), which are ROS, act as redox signaling substances at physiological levels and are often generated by more than 40 enzymes including NADPH oxidase and the mitochondrial electron transport chain under the control of growth factors and cytokines. However, when signaling and ROS levels persist for a long time, it leads to the accumulation of oxidative damage and metabolic disorders. Therefore, the present inventor decided to investigate whether the contemplated composition can counteract oxidative stress. In particular, the contemplated composition showed a significant decrease in reactive oxygen species, as detailed below, in addition to all of the beneficial functions described above.

[0091] Furthermore, since the contemplated composition is based on the components of the Mediterranean diet (known as a common denominator among long-lived people), the inventor also initiated an investigation into whether the composition presented herein affects aging-related genes. In the following exemplary results, significant downregulation of various aging-related genes was demonstrated.

[0092] Acetylcholinesterase (AChE) is an enzyme mainly present in blood and nerve synapses and plays an important role in various cognitive processes. AChE is a catalyst that hydrolyzes the neurotransmitter acetylcholine into choline and acetic acid, which is a necessary reaction for cholinergic neurons to return to a resting state after activation. AChE inhibition (e.g., donepezil, etc.) is an important target in the treatment of Alzheimer's disease, and AChE inhibitors are the most commonly used drugs for its treatment. In addition to Alzheimer's disease, AChE inhibitors are useful for the diagnosis and treatment of diseases such as glaucoma, myasthenia gravis, and bladder dilation. Therefore, AChE inhibitors are thought to have an effect of promoting cognition. For this purpose, the present inventors also determined whether the compositions introduced herein have an inhibitory effect on AChE. In particular, as shown in more detail below, the compositions presented herein have a substantial inhibitory effect on AChE, and as a result, among other advantages, there is a particular potential to significantly improve cognition.

[0093] In a further contemplated aspect of the subject matter of the present invention, the compositions presented herein can be formulated in various forms, and particularly preferred formulations include those combined with a nutritionally or pharmaceutically acceptable carrier and are most preferably formulated for oral administration (although parenteral administration is also explicitly contemplated). Therefore, the contemplated compositions can be formulated as solid or liquid products. For example, when the contemplated composition is formulated as a solid product, suitable product forms include single-dose unit formulations such as capsules, tablets, powders, etc., and other solid formulations include snack bars, gummies, or other edible products where the composition is coated (e.g., on cereal) or the composition is mixed or layered (e.g., in chewing gum). As another example, when the contemplated composition is formulated as a liquid product, suitable product forms include flavored and / or carbonated beverages (e.g., tea, juice), and / or functional beverages (e.g., sports drinks or energy drinks), or infusions, or liquid dairy products (e.g., yogurt, kefir).

[0094] Thus, the contemplated compositions may be provided together as part of a food or beverage product and / or provided in single-dose units for consumption. Most typically, the daily dosage of the contemplated composition (excluding the carrier) is preferably at least 10 mg, at least 50 mg, at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, at least 750 mg, at least 1,000 mg, or at least 1,500 mg. For example, suitable dosages are 10 - 50 mg, 50 - 100 mg, 100 - 200 mg, 200 - 400 mg, 300 - 600 mg, 400 - 800 mg, 600 - 1,000 mg, or 1,000 - 2,000 mg.

[0095] Most typically, the contemplated compositions are orally administered over a long period of at least one week, at least two weeks, at least four weeks, at least two months, at least three months, at least six months, or at least one year or more to maintain or support healthy aging. Administration is preferably daily, at least twice a week, or at least three times a week. In some embodiments, the contemplated compositions may be administered topically, generally in the form of a cream, lotion, essence elixir, or shampoo. In this case, the composition can be obtained as a clear solution as a (optionally filtered) aqueous or hydroalcoholic extract.

[0096] It will be readily understood that the contemplated compositions may also be combined with one or more additional ingredients to impart further desirable functionality. Suitable additional ingredients include vitamins (e.g., single vitamins or vitamin blends such as multivitamin blends), trace elements or minerals in food (e.g., individual elements or minerals, or multiple elements and minerals mixed in various forms), various special compounds and mixtures (e.g., prebiotics, human milk oligosaccharides), and / or one or more probiotic microorganisms (e.g., genus Lactobacillus, genus Bifidobacterium, genus Leuconostoc, Saccharomyces boulardii, etc.), postbiotics (e.g., short-chain fatty acids such as butyric acid, secondary bile acids such as (tauro)ursodeoxycholic acid), dietary fibers (e.g., water-soluble dietary fibers or insoluble dietary fibers), and / or nutritionally acceptable oligosaccharides (e.g., xylooligosaccharides (XOS) or fructooligosaccharides (FOS)). Similarly, the contemplated compositions may also contain niacin, niacinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and / or nutritionally acceptable CD38 inhibitors, thereby increasing intracellular NAD and / or NADP levels (as well as their respective reduced forms NADH and NADPH). Thus, the contemplated compositions can further (synergistically) enhance or support cell metabolism and energy, in particular.

[0097] Of course, it should be recognized that the compositions according to the subject matter of the present invention may be administered not only to humans but also to other non-human mammals, particularly livestock and companion animals (e.g., dogs, cats, horses). Administration is typically carried out once to three times a day (and in some cases even more frequently) over at least two days, three days, five days, one week, two to four weeks, one to three months, or even longer periods. Most typically, administration can be carried out prophylactically for a period sufficient to at least alleviate symptoms (e.g., pain and swelling associated with inflammation, reduced energy levels, frequent infections, etc.) or to help avoid or reduce the exacerbation of the health condition.

Examples

[0098] Typical composition: Unless otherwise indicated, all tests were carried out using a defined mixture of selected polyphenol-containing products / extracts common to the Mediterranean diet. The polyphenol-containing products / extracts were obtained from raw materials characterized by the following colors. Red group (sub-blend): apple extract, pomegranate extract, tomato powder, beetroot, green group (sub-blend): olive extract, rosemary extract, unroasted coffee bean extract, kale; orange / yellow group (sub-blend): onion extract, ginger extract, grapefruit extract, carrot; and, purple / blue group (sub-blend): grape extract, blueberry extract, blackcurrant, elderberry. Corn starch, silica, and sunflower lecithin were used as processing aids. The relative ratios are shown in Table 1 below.

Table 1

[0099] Phytochemical HPLC / MS / MS analysis: HPLC / MS composition analysis of the above exemplary compositions revealed the following raw materials and ratios. Each column in Tables 2 - 8 shows the analyte ID (column 1), chemical entity (column 2), M-H (column 3), RT (column 4), peak intensity (column 5), and MS / MS fragment (column 6).

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

[0100] Unless otherwise specified, the effects of representative compositions were tested for their regulatory effects on various target entities related to mild / chronic inflammation, age-related decline in immunity, energy metabolism, aging, reactive oxygen species, acetylcholinesterase activity, obesity, and viability, and exemplary activity results are shown below. Cytokines:

[0101] In the following experiments, the inventors conducted a series of tests to examine the effects of representative compositions and sub-blends on various cytokines. For this purpose, the tested compositions were used with LPS-treated primary human monocytes and SEB-induced T lymphocytes (T helper cells, CD4+) to measure specific responses regarding selected inflammatory cytokines. Unless otherwise stated, all test materials were provided as solid powders dissolved in water.

[0102] Here, the inventors used primary human monocytes / lymphocytes to test the anti-inflammatory effects of the test articles using established and published methods, which will be detailed below. Human monocytes are one of the main cell types involved in peripheral inflammation because they are the main cells affected by bacterial lipopolysaccharide (LPS) produced by Gram-negative bacteria. Furthermore, cytokine release from monocytes is also considered one of the first steps in the inflammatory cascade, and representative cytokines include inflammatory cytokines such as interleukin-1 (IL-1) beta, IL-6, IL-8, IL-23, tumor necrosis factor (TNF) alpha, prostaglandin E2 (PGE2), chemokines such as monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) and macrophage inflammatory protein-1 (MIP-1), and free radical and oxidative stress markers such as isoprostane. Monocytes can also trigger the inflammatory cascade in other cell types such as fibroblasts, while T cells respond to cytokine signaling and play a central role in the adaptive immune response.

[0103] T cells can be easily distinguished from other lymphocytes by the presence of T cell receptors (TCRs) on their cell surface. Interleukin-2 (IL-2) is a 15.5-16 kDa protein mainly produced by activated CD4+ T cells (T helper cells), and is produced, for example, by enterotoxin B type, an enterotoxin produced by Staphylococcus aureus, a Gram-positive bacterium, also known as staphylococcal enterotoxin B (SEB). IL-2 controls the activities of white blood cells (leukocytes, mostly lymphocytes) that govern immunity. IL-2 is part of the body's natural response to microbial infection and distinguishes foreign substances ("non-self") from "self". The investigated parameters are established inflammatory mediators regarding inflammation and are known to have an important role in the inflammatory cascade by inducing pain and inflammation.

[0104] Measurement of cytokines in primary human monocytes: Human primary monocytes are isolated (enriched) from the buffy coats of healthy human blood donors. For ELISA experiments, the cells are seeded in 24-well plates (about 500,000 cells / ml in 1 ml). The cells are cultured with LPS (10 ng / ml) for 24 hours. Representative compositions (5 doses) and dexamethasone as an anti-inflammatory control are added 30 minutes before LPS treatment (untreated cells served as negative controls). After 24 hours, the supernatants are removed, centrifuged, and the required inflammatory parameters such as MCP-1, IL-8, IL-6, IL-23, and TNF-alpha concentrations are examined according to the manufacturer's protocol using ELISA (MCP-1, TNF-alpha, IL-6, IL-23, and IL-8, Research and Development Department of Biotechne). Each dose was investigated 6 times with 2-3 buffy coats from 2-3 different donors (n = 2-3 per buffy coat, total n = 6).

[0105] Measurement of PGE2 and isoprostane (free radical marker) in primary human monocytes: Primary human monocytes were isolated (concentrated) from the meninges of healthy human blood donors. For the EIA experiment, the cells were seeded in 24-well plates (about 500,000 cells / ml in 1 ml). The cells were cultured with LPS (10 ng / ml) for 24 hours. Representative compositions (5 doses) and dexamethasone as an anti-inflammatory control were added 30 minutes before LPS treatment (untreated cells served as negative controls). After 24 hours, the supernatant was removed, centrifuged, and the concentrations of isoprostane and PGE2 were examined using EIA (manufactured by Cayman, distributed by Biomol, Hamburg, Germany). Each dose was investigated 6 times with 2 - 3 meninges from 3 different donors (n = 2 - 3 per meninges, total n = 6).

[0106] Measurement of IL-2 in primary T cells: Human primary T cells are isolated (concentrated) from the meninges of healthy human blood donors. For the ELISA experiment, the cells are seeded in 24-well plates (about 500,000 cells / ml in 1 ml). The cells are cultured with SEB (1 μg / ml) for 24 hours. Representative compositions (5 doses) and dexamethasone or hydrocortisone as an anti-inflammatory control are added 30 minutes before SEB treatment (untreated cells served as negative controls). After 24 hours, the supernatant is removed, centrifuged, and the IL-2 concentration is examined using ELISA (Biotechne, Wiesbaden, Germany) according to the manufacturer's protocol (other parameters are also possible). Each dose was investigated 6 times with 2 - 3 meninges from 2 - 3 different donors (n = 2 - 3 per meninges, total n = 6).

[0107] As can be seen from the data shown in FIGS. 1-7, the representative composition showed significant effects on various inflammatory markers in primary human monocytes treated with LPS. As shown in the summary of FIG. 1, the representative composition strongly and significantly inhibited the release of IL-6, TNF-alpha, and PGE2 induced by LPS, starting from a low dose of 10 μg / ml. The release of the free radical marker LPS-induced isoprostane (PGF2-alpha) was suppressed at high doses of the extract. LPS-induced IL-23 release was not affected, but the levels of IL-8 and MCP1 were slightly increased. LPS-mediated IL-1 beta release was enhanced by this composition. The anti-inflammatory drug and the positive control dexamethasone strongly blocked all parameters except IL-23. FIG. 2 shows the inhibitory effect of the representative composition on various LPS-induced inflammatory cytokines in primary human monocytes (*p<0.05, ***p<0.01, ***p<0.001 with respect to the LPS control (t-test)). More specifically, FIG. 3 shows the inhibitory effect of the representative composition on LPS-induced TNF-alpha release in primary human monocytes (*p<0.05, ***p<0.01, ***p<0.001 with respect to the LPS control (t-test)). FIG. 4 shows the inhibitory effect of the representative composition on LPS-induced IL-6 release in primary human monocytes (*p<0.05, ***p<0.01, ***p<0.001 with respect to the LPS control (t-test)). FIG. 5 shows the inhibitory effect of the representative composition on LPS-induced PGE2 release in primary human monocytes (*p<0.05, ***p<0.01, ***p<0.001 with respect to the LPS control (t-test)). FIG. 6 shows the inhibitory effect of the representative composition on LPS-induced isoprostane release in primary human monocytes (*p<0.05, ***p<0.01, ***p<0.001 with respect to the LPS control (t-test)).

[0108] As can be further seen from the data in Figure 7, the representative composition also showed a significant effect on IL-2 synthesis in primary human monocytes (primary human T cells) treated with SEB. Here, the representative composition significantly inhibited LPS-induced IL-2 at a high dose (e.g., 250 μg / ml). The anti-inflammatory drug and the positive control dexamethasone strongly inhibited IL-2 synthesis during the experiment as described above. Figure 7 shows the inhibitory effect of the representative composition on SEB-induced L-2 release in primary human lymphocytes (*p < 0.05, ***p < 0.01, ***p < 0.001 with respect to the SEB control (t-test)). Notably, while the representative composition is effective in blunting or reducing inflammatory signaling, it only moderately or not at all reduces T cell stimulation (generally required for antigen-specific immune responses).

[0109] To investigate the potential active fractions contained in the representative composition, the inventor then conducted the above-described experiments using only the specific sub-blends described above. For this purpose, the inventor tested the sub-blends of the red group, the green group, the orange / yellow group, and the purple / blue group, and the typical results regarding TNF-alpha, IL-6, and PGE2 are shown in Figures 8 - 11 (*p < 0.05, **p < 0.01, ***p < 0.001 with respect to the LPS control (t-test)). Figure 8 shows an overview of the inhibition data for the sub-blend of the green group, Figure 9 shows an overview of the inhibition data for the sub-blend of the red group, Figure 10 shows an overview of the inhibition data for the sub-blend of the orange / yellow group, and Figure 11 shows an overview of the inhibition data for the sub-blend of the purple / blue group.

[0110] As can be readily seen from the data in Figure 8, the inhibition of cytokine release of TNF-alpha and IL-6 using only the green sub-blend was moderate in a dose-dependent manner, but no obvious inhibition of PGE2 release was observed at all the concentrations tested. On the other hand, when the red sub-blend was used, as shown in Figure 9, no significant inhibition was observed for any of the cytokines tested. However, at high concentrations of the red sub-blend, IL-6 and TNF-alpha increased. Similarly, the orange / yellow sub-blend, as can be seen from Figure 10, did not result in significant inhibition or increase for any of the cytokines tested. As shown in Figure 11, only when the purple / blue sub-blend was used, a slight inhibition of TNF-alpha was observed.

[0111] Notably, as can be readily seen from Figure 12 (*p < 0.05, **p < 0.01, ***p < 0.001 with respect to the LPS control (t-test)), when the four sub-blends were used in combination, a strong inhibitory synergistic effect was observed with respect to TNF-alpha, IL-6, and PGE2. Such synergistic properties are not only unexpected, but strongly suggest that the combination with this synergistic effect is effective in reducing the signs and symptoms of various inflammatory conditions (subacute, chronic, disease-related, allergy-related, etc.) spanning multiple different inflammatory pathways. Such multi-pathway inhibition is considered to be significantly more effective than conventional anti-inflammatory drugs and to result in a physiologically tolerable anti-inflammatory immunomodulation. Furthermore, since all the components of the representative composition are derived from food, it is possible to beneficially achieve the reduction of inflammation in many symptoms without the side effects typical of conventional anti-inflammatory drugs (by targeting multiple different pathways).

[0112] To investigate whether the representative composition also affects other tissues (e.g., muscle cells), the inventor measured the mRNA expression of IL-6 and TNF-alpha in murine C2C12 muscle cells (an immortalized mouse myoblast cell line). The C2C12 cells were provided by the University of Cordoba, Cordoba, Spain. The cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), 4.5 mM L-glutamine, and 1% penicillin and streptomycin antibiotics. In all experiments, the cells were grown to 80-90% confluence in 24-well plates.

[0113] RNA isolation and quantitative PCR: Cultured cells were incubated for 4 hours with various concentrations of the representative composition dissolved in water (1-100 μg / ml). Total RNA was extracted using the Universal RNA Kit (roboklon). cDNA synthesis was performed by reverse transcription from 1 μg of total RNA using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Mannheim, Germany), RNase Inhibitor rRNasin® (Promega, Mannheim, Germany), dNTP master mix (Promega, Mannheim, Germany), and random hexamer primers (Biomers, Germany). Real-time PCR amplification was performed using ORA TM qPCR Green ROX H Mix, 2X on Analytik Jena's qTOWER 2.0 / 2.2 Quantitative Real-Time PCR Thermal Cyclers. The reaction conditions were 40 cycles of 3 minutes at 95°C, 15 seconds at 95°C, 30 seconds at 50°C, and 45 seconds at 72°C, with plate readings taken every cycle. Subsequently, a melt curve condition of 1 minute at 95°C, 1 minute at 55°C, and then from 65°C to 95°C in 0.5°C increments for 5 seconds was performed, followed by a final plate reading. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) functioned as an internal control for sample normalization, and the comparative cycle threshold Ct method was used for data quantification.

[0114] The effects of representative compositions on IL-6 and TNF-alpha mRNA levels in C2C12 muscle cells are shown in the exemplary results of FIGS. 13 and 14 (both *p<0.05, **p<0.01, ***p<0.001). As readily apparent, the representative composition potently and dose-dependently decreased IL-6 and TNF-alpha mRNA levels starting at a dose of 10 μg / ml. Thus, it will be understood that the representative composition has a major impact on the synthesis and secretion of the selected cytokines. NFκB:

[0115] In the following experiments, since the present inventor considered NF-κB to be one of the most important regulatory factors for pro-inflammatory gene expression, a series of experiments were conducted to examine the effect of the representative composition on NFκB. For example, the synthesis of various cytokines such as TNF-alpha, IL-1 beta, IL-6, and IL-8 is mediated by NF-κB, similar to the expression of cyclooxygenase 2 (Cox-2).

[0116] To measure NF-kappa B transcriptional activity, the present inventor used an assay kit that utilized HEK293t cells expressing NF-κB (nuclear factor kappa-light-chain enhancer of activated B cells) and containing a luciferase reporter gene functionally linked to an upstream NF-κB genetic response element. Thus, by quantifying changes in luciferase expression, changes in NF-κB activation levels can be sensitively substituted. NF-κB is a signal transduction-dependent transcription factor. This NF-κB reporter cell line has been verified to show a strong dose-dependent activation response when treated with TNFα or phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator. Therefore, this assay is particularly suitable for screening test samples for functional activities that may induce or suppress NF-κB activity. Here, the NF-kappa B transcriptional activity assay was performed according to the manufacturer's instructions (an assay commercially available from INDIGO Biosciences (Biomol)).

[0117] The effects of the representative composition were observed using the above assay method. As is clear from the data in Fig. 15 (*p<0.05, **p<0.01, ***p<0.001), the PMA-induced activation of NF-κB is enhanced at low doses (1 and 10 μg / ml, which are thought to be artifacts of the test system), but at high doses, starting from a dose of 50 μg / ml and using extracts of 100 μg / ml and 250 μg / ml, the activation of NF-κB was strongly and dose-dependently almost completely inhibited. In particular, this inhibition was more potent than the known NF-κB inhibitor QNZ. This inhibition of NF-κB was also consistent with the inhibition observed across various inflammatory cytokines and pathways, as shown above. Glucose uptake:

[0118] In the following experiments, the inventor conducted a series of assays to determine the effect of the representative composition on energy metabolism, particularly glucose uptake.

[0119] For this purpose, C2C12 cells (5x10 4 ) were seeded in a 96-well black plate and cultured for 24 hours. Then, the medium was removed, the cells were cultured in OptiMEM, labeled with 50 μM of 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose), and treated with the representative composition or the positive control rosiglitazone for 24 hours. The medium was removed, the wells were carefully washed with PBS, and cultured with PBS (100 μl / well). Finally, fluorescence was measured according to the manufacturer's instructions.

[0120] Figure 16 shows exemplary results regarding the effect of a representative composition on glucose uptake in mouse C2C12 muscle cells (*p < 0.05, **p < 0.01, ***p < 0.001). As can be readily seen from the graph data, the effect of the representative composition on glucose uptake was very high and, in fact, had a potency comparable to that of rosiglitazone, a known antidiabetic compound. Thus, it will be understood that the representative composition had a significant antidiabetic therapeutic effect and substantially promoted cellular energy. Mitochondrial biogenesis:

[0121] Since the available energy substrates were significantly increased, the inventors conducted a series of experiments to examine whether the increased energy metabolites could be effectively utilized in glycolysis and cellular respiration. Since mitochondrial activity is required for cellular respiration, the inventors attempted to examine the effect of the representative composition on mitochondrial biogenesis in nerve cells.

[0122] For this purpose, Neuro-2a (N2a) (ATCC, Manassas, Virginia, USA) cells were cultured in DMEM supplemented with 10% FBS, 2 mM l-glutamine, and 1% penicillin / streptomycin. The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

[0123] Determination of mitochondrial biogenesis. N2a cells were seeded in 96-well plates (3.5 × 10 3 cells per well) and, after 24 hours, stimulated for 72 hours in triplicate wells with increasing concentrations of the representative composition (5 doses). Thereafter, Mitotracker Green (100 nM; Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to the medium for 30 minutes. The cells were washed with PBS and fresh medium was added. Images were taken and fluorescence was measured using the cell imaging system IncuCyte HD (Essen BioScience, Hertfordshire, UK). Rosiglitazone was used as a positive control.

[0124] As can be easily seen from the results of Fig. 17, the representative composition significantly increased mitochondrial biogenesis. Surprisingly, mitochondrial biogenesis using the representative composition exceeded that of rosiglitazone, which is the positive control in this assay. ATP:

[0125] In the following experiments, the inventors conducted a series of tests to examine the effect of the representative composition on various intracellular ATP levels. For this purpose, the inventors used macrophages of RAW264.1 mice and muscle cells of C2C12 mice (obtained from the University of Freiburg, Germany).

[0126] The cells were maintained in DMEM medium (DMEM complete medium) containing 10% FBS and 1% antibiotic penicillin / streptomycin at 37 °C in a humidified atmosphere of 5% CO2. In the ATP assay, RAW cells and C2C12 were seeded at a density of 2x10 4 cells / well in a 96-well plate and cultured overnight at 37 °C in a humidified atmosphere of 5% CO2 in DMEM medium. Next, the cell cultures were stimulated with the representative composition at selected concentrations (5 doses, n = 4). ATP in the cells was measured using the CellTiter-Glo(R) 2.0 Cell Viability / ATP Assay.

[0127] Specifically, in macrophages, only the low dose of 1 μg / ml of the representative composition showed a moderate increasing effect on ATP levels, as shown in Fig. 18 (*p < 0.05, **p < 0.01, ***p < 0.001). There was no effect at other doses. In C2C12 cells, the representative composition inhibited the ATP level in a dose-dependent manner although weakly (see Fig. 19: *p < 0.05, **p < 0.01, ***p < 0.001). Reactive oxygen species (ROS):

[0128] In the following experiments, the inventors conducted a series of experiments to determine the effect of the representative composition on the antioxidant capacity of cells by determining reactive oxygen species (ROS) in a macrophage model system.

[0129] For this purpose, the inventors used macrophages of RAW264.1 mice obtained from the Uniklinik Freiburg in Germany. The cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in DMEM medium (DMEM complete medium) after addition containing 10% FBS and 1% antibiotic penicillin / streptomycin. The cell reaction was examined in the presence of representative compositions and controls (no compound as negative control, trolox and vitamin C as positive controls). As is clear from the results in Figure 19, the ROS levels induced by H2O2 were strongly reduced by the representative compositions starting from a relatively low dose of 1 μg / ml. Notably, the maximum ROS suppression was achieved with 50 μg / ml of DC, which is comparable in efficacy to trolox C, one of the most potent and commonly used antioxidants. Adipokine:

[0130] In the following experiments, the inventors conducted a series of experiments to examine the effect of the representative composition on various adipokines associated with obesity and metabolic diseases. More specifically, the inventors used 3T3-L1 cells (preadipocyte fibroblasts) in a standard cell culture model and measured the expression levels of an extended set of adipokines using a proteome profiler kit.

[0131] Low passage 3T3-L1 cells (ATCC) were cultured in DMEM supplemented with 10% (v / v) fetal bovine serum (referred to as DMEM / FBS), 25 mM glucose, 4 mM L-glutamine, and 1 mM sodium pyruvate. Cells were cultured in 12-well plates in a humidified incubator at 37 °C with 5% (v / v) CO2. Two days after confluence, differentiation into adipocytes was induced with a cocktail of stimulants including 2 μg / mL insulin, 0.5 mM isobutylmethylxanthine, 0.25 μM dexamethasone, and 2 μM rosiglitazone (all purchased from Sigma) in DMEM / FBS. Three days later, the differentiation medium was removed and the cells were maintained in post-differentiation medium (DMEM / FBS supplemented with 2 μg mL-1 insulin) for an additional 6 - 9 days, with medium changes every two days or as needed. Differentiation was observed daily by microscopy.

[0132] After differentiation, cells were washed with PBS and cultured in complete DMEM medium and treated with TNFα (50 ng / ml) for 24 h in the absence and presence of representative compositions (10 μg / ml and 100 μg / ml, dissolved in water). Supernatants were collected and assayed to detect adipokines and other related soluble mediators. Adipokines and other obesity-related proteins were detected using a semi-quantitative proteome profiler mouse adipokine array kit (R&D System, Minneapolis, MN, USA). Pixel densities of developed X-ray films were collected using a scanner and analyzed using the processing and analysis program ImageJ (NIH, Bethesda, MD, USA).

[0133] The following adipokines and soluble mediators were simultaneously monitored: adiponectin, AgRP, ANGPT-L3, C-reactive protein, DPPIV, endocan, fetuin A, acidic FGF, FGF-21, HGF, ICAM-1, IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IL-6, IL-10, IL-11, leptin, LIF, lipocalin-2, MCP-1, M-CSF, oncostatin M, pentraxin 2, pentraxin 3, Pref-1, RAGE, RANTES, RBP4, resistin, serpin E1, TIMP-1, TNF-alpha, VEGF.

[0134] Figures 19 to 22 show the selected effects of representative compositions on the secretion of obesity-related proteins in differentiated 3T3-L1 cells. Most notably, TNFα induced the secretion of IGFBP-1, leptin, oncostatin M and resistin, and this secretion was completely inhibited by the representative composition as shown in Figure 21. On the other hand, as shown in Figures 22 and 23, ANGPT-L3, C-reactive protein, endocan, FGF-21, HGF, IGFBP-2, IL-11, RBP4 and pentraxin 2 were clearly induced by the TNFα + representative composition but not by TNFα alone, suggesting that the synthesis of these proteins was induced by the representative composition. Aging:

[0135] In the following experiments, the inventor conducted a series of experiments to examine the effect of a representative composition on the gene expression of selected aging markers in normal human dermal fibroblasts (NHDF).

[0136] NHDF cells were purchased from ATCC in Germany. The NHDF cells were cultured and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS), 4.5 mM L-glutamine, and 1% penicillin and streptomycin antibiotics. The NHDF cells were cultured at 37 °C under 5% CO2 humidification. In all experiments, the cells were grown to 80 - 90% confluence in 24-well plates. The medium was changed 24 hours after cell culture, and the NHDF cells were stored for 6 days to age them.

[0137] Quantitative real-time PCR (qPCR) was performed using senescent normal human dermal fibroblasts. The cultured senescent NHDF cells were incubated for 4 hours with various concentrations of representative compositions dissolved in water (1 - 100 μg / ml). Total RNA was then extracted using the Universal RNA Kit (roboklon). cDNA synthesis was reverse transcribed from 1 μg of total RNA using Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase (Promega, Mannheim, Germany), RNase Inhibitor rRNasin® (Promega, Mannheim, Germany), dNTP master mix (Promega, Mannheim, Germany), and random hexamer primers (Biomers, Germany). Real-time PCR amplification was performed using the ORA TM qPCR Green ROX H Mix, 2X on the qTOWER 2.0 / 2.2 Quantitative Real-Time PCR Thermal Cyclers from Analytik Jena. HGF, c-fos, p16 INK、Primers were designed to amplify the gene encoding p21. The reaction conditions were as follows: a cycle of 95°C for 3 minutes, 95°C for 15 seconds, 50°C for 30 seconds, and 72°C for 45 seconds was performed 40 times, and plate readings were taken for each cycle. Subsequently, it was carried out under melt curve conditions of 95°C for 1 minute, 55°C for 1 minute, 65°C, and 95°C with a 0.5°C increment every 5 seconds, and the final plate reading was taken. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) functioned as an internal control for sample normalization, and the comparative cycle threshold Ct method was used for data quantification.

[0138] Of note, as can be seen from Figure 24, representative compositions strongly inhibited the expression of the aging-related genes HGF, c-fos, p16 INK and p21 even at very low doses. Acetylcholinesterase (AChE):

[0139] In the following experiments, the inventor conducted a series of experiments to examine the effect of representative compositions on AChE as a potential target related to nerve function.

[0140] For this purpose, the inventor used AmpliteTM Colorimetric assay (manufactured by AAT Bioquest) according to the manufacturer's instructions. 40 μl of assay buffer for NIC (no inhibition control added), 40 μl of AHE solution, and 40 μl of donepezil stock solution diluted with assay buffer as a negative control were pipetted into separate wells of a 96-well plate. For the negative control, NICs, and test sample solutions, 10 μl of acetylcholinesterase solution (4 mU / 10 μl) was added to each well. Control samples and samples with a representative composition were gently shaken and cultured at room temperature for 15 minutes. 50 μL of AChE working solution was added to each well, and the total assay amount of acetylcholinesterase was set to 100 μl / well (t = 0 min). The reaction mixture was incubated at room temperature for 60 minutes in the dark (t = 60 min). Absorbance was measured at 405 nm using a PerkinElmer Victor X5 2030-0050 Multimode Plate Reader from PerkinElmer (Rotgau, Germany) at time points t = 0 min and t = 60 min.

[0141] In Figure 25 showing the effect of a representative composition on the acetylcholinesterase activity, exemplary test results are shown. The results are expressed as relative units ± SD (*p < 0.05, **p < 0.01, ***p < 0.001). As can be easily seen, the representative composition showed a significant AChE inhibitory effect at high concentrations. Survival rate:

[0142] In the following experiment, the inventor conducted a series of experiments to examine the effect of a representative composition on the cell survival rate of macrophages. Here, RAW264.1 mouse macrophages were obtained from the Uniklinik of Freiburg and maintained in DMEM medium (DMEM complete medium) containing 10% FBS and 1% antibiotic penicillin / streptomycin at 37 °C in a humidified atmosphere of 5% CO2.

[0143] The cytotoxicity assay was performed by plating RAW cells at 2x10 4Seeding was performed at a density of cells per well and cultured overnight at 37 °C in a humidified atmosphere of 5% CO2 in DMEM medium. Next, cell culture was stimulated with representative compositions at selected concentrations (7 doses, n = 4), and cell viability was measured by Alamar blue staining using NaF as a toxicity control. Exemplary results are shown in Figure 26. As can be readily seen from the results, only high doses of 500 μg / ml or more showed any effect on cell viability and cell metabolism, while no statistically significant effect on cell viability and cell metabolism was observed at low concentrations.

[0144] Further aspects, considerations, and intentions suitable for use in this specification are disclosed in U.S. Patent No. 11,065,295 and are incorporated herein by reference.

[0145] In some embodiments, it is to be understood that numerical values representing amounts of raw materials, properties such as concentrations and reaction conditions, used in the description and claims of certain embodiments of the present invention are in some cases modified by the term "about". Accordingly, in some embodiments, the numerical parameters recited in the description and appended claims are approximate values that may vary depending on the properties obtained by a particular embodiment. The recitation of a range of values herein is merely intended to function as a concise method of individually referring to each separate value falling within that range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0146] As used herein, the term "administering" a pharmaceutical composition or a nutritional supplement composition refers to both direct and indirect administration of the pharmaceutical composition or the nutritional supplement composition. Direct administration of the pharmaceutical composition or the nutritional supplement composition is generally performed by a medical professional (e.g., a physician, a nurse, a dietitian, etc.). Indirect administration includes steps of providing or making available the pharmaceutical composition or the nutritional supplement composition to a medical professional or an individual in need thereof for direct administration (e.g., injection, infusion, oral administration, topical administration, etc.). Further, it should be noted that the terms "prognosing" or "predicting" a medical condition, susceptibility, or response to an intended treatment mean including the act or prediction of predicting (but not treating or diagnosing) a medical condition, susceptibility, and / or response, including the rate of progression, improvement, and / or persistence of a disease in a subject.

[0147] All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly inconsistent with the context. The use of any and all examples, or exemplary language (e.g., "such as") for particular embodiments provided herein is merely intended to better clarify the invention and does not limit the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0148] As used in this specification and the following claims, unless the context clearly dictates otherwise, the meanings of "a", "an", and "the" include plural references. Also, as used herein, the meaning of "in" includes "in" and "on", unless the context clearly dictates otherwise. As used herein, and unless otherwise indicated by context, the term "coupled to" is intended to include both direct coupling (where two elements that are coupled together are in contact with each other) and indirect coupling (where at least one additional element is located between the two elements). Accordingly, "coupled to" and "coupled with" are used as synonyms.

[0149] It will be apparent to those skilled in the art that many variations, in addition to those already described, are possible without departing from the inventive concepts herein. Accordingly, the subject matter of the present invention is not limited except as by the appended claims. Further, in interpreting both the specification and the claims, all terms should be interpreted as broadly as possible consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the recited elements, components, or steps may be present, utilized, or combined with other elements, components, or steps not expressly recited. Where reference is made in the specification or claims to at least one selected from the group consisting of A, B, C... and N, the text should be interpreted as requiring only one element from the group, rather than A plus N, B plus N, etc.

Claims

1. A nutritionally acceptable carrier combined with a plurality of chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purple-blue colors, wherein the red plant materials include apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant materials include olive extract, rosemary extract, unroasted coffee bean extract, and kale powder, the orange-yellow plant materials include onion extract, ginger extract, grapefruit extract, and carrot powder, and the purple-blue plant materials include grape extract, blueberry extract, bilberry powder, and elderberry powder, and the red plant materials, the green plant materials, the orange-yellow plant materials, and the purple-blue plant materials are present in synergistic amounts with respect to reducing the release of at least one of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE2), and isoprostane, and the nutritional composition is formulated as a capsule, gummy, or powder for oral administration.

2. The composition according to claim 1, wherein the composition inhibits the activation of NFκB.

3. The composition according to claim 1, wherein the composition increases the uptake of glucose into cells.

4. The composition according to claim 1, wherein the composition reduces oxidative damage by reactive oxygen species.

5. The composition according to claim 1, wherein the composition reduces the expression of inflammatory adipokines.

6. The composition according to claim 1, wherein the composition is formulated in single-dose units.

7. The composition according to claim 6, wherein the single-dose unit contains 50 to 1000 mg of the composition.

8. The composition according to claim 1, further comprising vitamins, trace elements or minerals in food, probiotics, and / or prebiotics.

9. The composition according to claim 1, further comprising niacin, niacinamide, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and / or a nutritionally acceptable CD38 inhibitor.

10. The composition according to claim 1, wherein the composition is administered for at least 30 days.

11. The composition according to claim 1, wherein the red plant material, the green plant material, the orange-yellow plant material, and the purple-blue plant material are part of the Mediterranean diet.

12. HGF, c-fos, p16 ＩＮＫ The composition according to claim 1, wherein the expression of an aging-related gene selected from the group consisting of, and p21 is suppressed.

13. The composition according to claim 1, wherein the composition increases intracellular ATP.

14. The composition according to claim 3, wherein the composition increases glucose uptake into cells to an extent equal to or greater than the increase in glucose uptake when rosiglitazone is used.

15. The composition according to claim 13, wherein the cell is a muscle cell.

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