CN117858630A

CN117858630A - Compositions and methods for combating processes associated with inflammation and aging and supporting cellular energy and/or metabolism

Info

Publication number: CN117858630A
Application number: CN202280057212.6A
Authority: CN
Inventors: 哈特利·庞德
Original assignee: Ricai Health Co ltd
Current assignee: Ricai Health Co ltd
Priority date: 2021-06-28
Filing date: 2022-06-17
Publication date: 2024-04-09

Abstract

Contemplated compositions and methods are based on a combination of polyphenols common to the Mediterranean diet, which, when combined, reduce the expression of pro-inflammatory signals and senescence-associated genes, and support mitochondrial biogenesis and cellular energy metabolism. Notably, the observed pleiotropic effects not only span multiple signaling pathways, but also exhibit synergistic activity in relation to a number of markers that reduce age-related energy decline, reduce age-related immune decline, and reduce age-related increase or persistence of chronic subacute inflammation. From a different perspective, contemplated compositions represent a harmonious combination of multiple biochemical molecules that form the basis of numerous benefits common in the mediterranean diet.

Description

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

The present application claims priority from U.S. provisional application Ser. Nos. 63/215,716, filed on 6/28 of 2021, and 63/285,591 filed on 12/3 of 2021, both of which are incorporated herein by reference.

Technical Field

The field of the present invention is compositions and methods for nutritional supplements, in particular to polyphenols and polyphenol mixtures and their use in the alleviation of various conditions associated with aging, such as mild/chronic inflammation, age-related reduced immunity, reduced energy metabolism and/or obesity.

Background

The background description includes information that may be helpful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

All publications and patent applications herein 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 usage of a term incorporated by reference does not conform to or is contrary to the definition of that term provided herein, then the definition of that term provided herein applies and the definition of that term as cited does not apply

Vitamins and other isolated nutritional compounds are diverse in species, and the benefits of these compounds are said to include immune support, anti-inflammatory effects, anti-aging effects, cardiac support, and digestive support, among others. Unfortunately, there is relatively little evidence that certain aspects of these so-called benefits are present when these vitamins and other isolated nutritional compounds are ingested. Also, if the nutritional supplement is in the form of an extract or powder of plant parts, various benefits will be advertised, but the actual benefits tend to be poor, or even not demonstrated at all. Furthermore, isolated nutritional compounds, as well as plant extracts and concentrates alone, often do not reflect a healthy diet.

Notably, certain geographic and ethnic diet types are associated with overall health, longevity, and/or physical recovery, and these beneficial effects are indeed well documented and proven. For example, the Mediterranean diet is generally associated with lower cardiovascular risk factors (see, e.g., nutrients 2018,10,379; doi:10.3390/nu 10030379), lower inflammatory and metabolic biomarkers, reduced risk of Alzheimer's disease (see, e.g., J Alzheimer's Dis.2010;22 (2): 483-492.), and reduced of certain inflammatory markers (see, e.g., nutrients 2018,10,62; doi:10.3390/nu 10010062). One common class of ingredients found in such diets are polyphenols, and various studies have been published regarding specific benefits 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.technology 2020.11: 10.1-10.38). However, due to the complexity and number of chemically diverse polyphenols, many studies have focused on only specific biochemical actions of individual polyphenols and such compounds, or provide general epidemiological information, without having molecular features without a more detailed diet.

In an effort to supplement the diet with various polyphenols, various supplements are known. For example, the Vital reactant (Gundry MD) provides a commercially available concentrated polyphenol powder mixture made from a variety of red plant materials to increase energy and promote digestion. Such mixtures advantageously comprise a plurality of chemically different polyphenols. However, the choice of plant material for use as a source of polyphenols does not reflect conventional dietary intake. Similarly, the mixture of the commercially available grape extract, olive extract, pomegranate extract, green tea extract, grapefruit extract, blueberry extract and orange extract sold by Fytexia are provided as antioxidant agents to protect cells from oxidative stress (see, e.g., the oxygenia sold by Fytexia). While the source components of such antioxidant formulations are beneficial in reducing oxidative stress, it has not generally been demonstrated to affect molecular signaling associated with (chronic subacute) inflammation, nfkb signaling, reduced energy metabolism, aging, age-related immune decline, and/or obesity.

Thus, while various nutritional supplements are known in the art, all or almost all of them suffer from various drawbacks. Accordingly, there is a need for compositions and methods that provide improved nutritional supplements, particularly those that have been demonstrated to combat (chronic subacute) inflammation, nfkb signaling, reduced energy metabolism, aging, age-related immune decline, and/or obesity.

Disclosure of Invention

The present inventors have now discovered various compositions and methods of specific combinations of polyphenols and/or polyphenols-rich substances (e.g., extracts and powders) common in food products of the Mediterranean diet that beneficially modulate various molecular characteristics associated with age-related reduced immunity, inflammation, reduced energy metabolism, and/or obesity. Thus, the polyphenols and/or polyphenol-enriched materials presented herein may be advantageously used in a nutritional-based approach to combat inflammation, which may be characterized by one or more of age-related reduced immunity, increased or chronic inflammation, reduced energy metabolism, and/or obesity.

In one aspect of the inventive subject matter, the inventors contemplate a nutritional composition comprising a nutritionally acceptable carrier in combination with a plurality of chemically different polyphenol containing plant materials having red, green, orange and violet colors, and in particularly preferred embodiments, the red, green, orange and violet plant materials have a synergistic effect in reducing proinflammatory cytokine release in human leukocytes.

For example, the red plant material may include apple extract, pomegranate extract, tomato powder, and beet root powder, the green plant material may include olive extract, rosemary extract, green coffee bean extract, and collard powder, the orange plant material may include onion extract, ginger extract, grapefruit extract, and carrot powder, and/or the purple blue plant material may include grape extract, blueberry extract, currant powder, and elderberry powder. From another perspective, the colored plant material may be selected on the basis of, or may be part of, a Mediterranean diet.

In some embodiments, the release of the pro-inflammatory cytokine is the release of at least one, or at least two, or at least three of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE 2), and isoprostadin. In other embodiments, the release of the pro-inflammatory cytokine is the release of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE 2), and isoprostadins.

It is further recognized that the compositions provided herein may also reduce nfkb expression, increase glucose uptake by cells, increase mitochondrial biogenesis in cells, reduce oxidative damage caused by reactive oxygen species, reduce expression of pro-inflammatory adipokines, and/or reduce expression of one or more senescence-associated genes.

Most typically, but not necessarily, the compositions will be formulated for oral administration, either as bulk materials or in single dosage units. For example, a single dosage unit may contain 50mg to 1000mg of the composition and may be formulated as a capsule, a jelly or a bulk powder. It will also be readily appreciated that contemplated compositions may also include vitamins, dietary trace elements or minerals, probiotics, and/or prebiotics. Similarly, contemplated compositions may also include niacin, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and/or a nutritionally acceptable CD38 inhibitor.

Thus, contemplated compositions are considered effective in treating or alleviating symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress.

Accordingly, the present inventors contemplate a method of supporting the health of a subject, wherein a composition as described herein is administered to an individual. For example, the composition may be administered in an amount effective to treat or reduce symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress. Thus, administration may last at least 30 days and/or be administered in daily doses of 50mg to 1000 mg. Thus, contemplated compositions are useful for supporting the health of a subject and healthy aging by orally administering the composition.

Thus, in a further aspect of the inventive subject matter, the inventors also contemplate a method of reducing inflammation in a subject, the 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 violet-blue colors, wherein the combination of plant materials reduces release of at least one pro-inflammatory cytokine in human cells, reduces expression of at least one senescence-associated gene in human cells.

In certain embodiments, the red plant material may include apple extract, pomegranate extract, tomato powder, and beetroot powder, the green plant material may include olive extract, rosemary extract, green coffee bean extract, and collard powder, the orange plant material may include onion extract, ginger extract, grapefruit extract, and carrot powder, and/or the violet plant material may include grape extract, blueberry extract, currant powder, and elderberry powder. Thus, the red, green, orange and violet plant materials are part of the mediterranean diet or may be selected based on the mediterranean diet.

Most typically, the pro-inflammatory cytokine is selected from the group consisting of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E2 (PGE 2), and isoprostadins. In particularly preferred aspects, the red, green, orange and violet plant materials will be present in synergistic amounts that reduce the release of pro-inflammatory cytokines in human cells. . As for the senescence-associated gene, it can be expected that the senescence-associated gene may be selected from HGF, c-fos, p16.sup.INK and p21.

Advantageously, the reduction of inflammation will include a reduction in age-related immunity, a reduction in age-related energy metabolism, a reduction in age-related mitochondrial biogenesis, a reduction in at least one symptom associated with inflammation, and/or a reduction in at least one symptom associated with metabolic syndrome.

Furthermore, it should be appreciated that in at least some embodiments, administration of the composition also advantageously reduces expression of nfkb, increases glucose uptake into cells, increases mitochondrial biogenesis in cells, reduces oxidative damage caused by reactive oxygen species, reduces expression of pro-inflammatory adipokines, and/or increases intracellular ATP.

Preferably, but not necessarily, the administration to the subject includes oral administration of plant material containing a plurality of chemically different polyphenols, e.g., at a dose of about 50mg to 1000mg. If desired, a variety of chemically unique polyphenol containing plant materials can be formulated into tablets, drinks or soft sweets. Furthermore, contemplated materials may also include vitamins, dietary trace elements or minerals, probiotics, and/or prebiotics. Likewise, the combination of plant materials may also comprise niacin, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and/or a nutritionally acceptable CD38 inhibitor.

In another aspect of the inventive subject matter, the inventors also contemplate a method of reducing inflammation in a subject, the method comprising the step of administering to the subject a nutritionally acceptable carrier in combination with a plant material having red, green, orange and violet colors comprising a plurality of chemically different polyphenols. Preferably, the red plant material comprises apple extract, pomegranate extract, tomato powder and beet root powder, the green plant material comprises olive extract, rosemary extract, green coffee bean extract and kale powder, the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder, and/or the violet plant material comprises grape extract, blueberry extract, currant powder and elderberry powder. In such methods, the combination of plant materials will reduce at least one pro-inflammatory cytokine, reduce nfκf signaling, and/or reduce at least one pro-inflammatory adipokine in the subject after administration. Preferably, but not necessarily, the combination of plant materials is a synergistic combination with respect to at least one cytokine reduction.

In another aspect of the inventive subject matter, the inventors contemplate a method of increasing glucose absorption by a cell comprising the step of contacting the cell with a plant material comprising a plurality of chemically distinct polyphenols having red, green, orange and violet colors, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract and carrot powder, and wherein the violet plant material comprises grape extract, blueberry extract, currant powder and elderberry powder. In this method, the combination of plant materials increases glucose uptake by cells after contacting the cells. Notably, as described in more detail below, an increase in glucose uptake by the cells is observed that is similar to or greater than (under otherwise identical conditions) an increase in glucose uptake with a thiazolinedione drug, such as rosiglitazone.

In a further aspect of the inventive subject matter, the inventors also contemplate a method of increasing ATP levels in a cell comprising the step of contacting the cell with a plant material comprising a plurality of chemically distinct polyphenols having red, green, orange and violet colors, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract and carrot powder, and wherein the violet plant material comprises grape extract, blueberry extract, currant powder and elderberry powder. In this method, the combination of plant materials increases the ATP level in the cells (particularly muscle cells) after contacting the cells.

Thus, from a different perspective, the inventors contemplate plant material having red, green, orange and violet colors comprising a plurality of chemically distinct polyphenols for alleviating at least one symptom of inflammation in an individual ingesting the plant material.

Similarly, the inventors also contemplate use of the nutritional compositions described herein to reduce the release of pro-inflammatory cytokines in human leukocytes, reduce nfkb signaling, increase glucose uptake by cells, increase mitochondrial biogenesis in cells, reduce oxidative damage caused by reactive oxygen species, reduce the expression of pro-inflammatory adipokines, and/or reduce the expression of at least one senescence-associated gene.

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

Drawings

FIG. 1 is a graph illustrating exemplary results of the effect of a representative composition on various cytokines in primary human monocytes.

FIG. 2 is a graph illustrating exemplary results of the effect of a representative composition on selected cytokines in primary human monocytes.

FIG. 3 is a graph illustrating exemplary results of the effect of a representative composition on TFN-alpha release in primary human monocytes.

FIG. 4 is a graph illustrating exemplary results of the effect of a representative composition on IL-6 release in primary human monocytes.

FIG. 5 is a graph illustrating PGE in representative compositions versus primary human monocytes ₂ Exemplary results of the influence of ReleaseA drawing.

Fig. 6 is a graph illustrating exemplary results of the effect of a representative composition on the release of isoprostadins from primary human monocytes.

FIG. 7 is a graph of exemplary results illustrating the effect of a representative composition on IL-2 synthesis in human primary lymphocytes.

FIG. 8 is a graph of exemplary results illustrating the effect of green sub-mixtures of representative compositions on selected cytokines in primary human monocytes.

FIG. 9 is a graph of exemplary results illustrating the effect of red sub-mixtures of representative compositions on selected cytokines in primary human monocytes.

FIG. 10 is a graph of exemplary results illustrating the effect of orange/yellow sub-mixtures of representative compositions on selected cytokines in primary human monocytes.

FIG. 11 is a graph of exemplary results illustrating the effect of purple/blue sub-mixtures of representative compositions on selected cytokines in primary human monocytes.

FIG. 12 is a graph illustrating exemplary results of the effect of a combined sub-mixture (representative composition) on selected cytokines in primary human monocytes.

FIG. 13 is a graph of exemplary results illustrating the effect of a representative composition on the expression of IL-6mRNA in C2C12 cells.

FIG. 14 is a graph of exemplary results illustrating the effect of representative compositions on TNF- α expression in C2C12 cells.

FIG. 15 is a graph of exemplary results illustrating the effect of a representative composition on NFkB expression in a HEK293 model system.

FIG. 16 is a graph illustrating exemplary results of the effect of a representative composition on glucose uptake by C2C12 muscle cells.

Fig. 17 is a graph illustrating exemplary results of the effect of representative compositions on mitochondrial biogenesis.

Fig. 18 is a graph illustrating exemplary results of the effect of representative compositions on mouse RAW 264.1 macrophage ATP levels.

FIG. 19 is a graph illustrating exemplary results of the effect of representative compositions on the level of ATP in C2C12 muscle cells.

Fig. 20 is a graph illustrating exemplary results of the effect of a representative composition on Reactive Oxygen Species (ROS) in mouse macrophages.

Fig. 21 is a graph illustrating exemplary results of inhibition of selected fat releasing hormone by representative compositions.

Fig. 22 is a graph illustrating exemplary results of stimulation of a first set of selected fat releasing hormones by a representative composition.

Fig. 23 is a graph illustrating exemplary results of inhibition of a second set of selected fat releasing hormones by a representative composition.

FIG. 24 is a graph illustrating exemplary results of the effect of a representative composition on gene expression of selected genes associated with senescence.

FIG. 25 is a graph illustrating exemplary results of the effect of representative compositions on acetylcholinesterase activity.

FIG. 26 is a graph of exemplary results illustrating the effect of representative compositions on macrophage cell viability and metabolism.

Detailed Description

The inventors have now found that a specific combination of polyphenol-containing materials (and polyphenols found therein) can strongly modulate many biomarkers associated with increased or chronic inflammation, nfkb signaling, reduced energy metabolism, aging, age-related immunity decline and/or obesity. Notably, this combination closely tracks the components and polyphenol-containing materials found in the Mediterranean diet (and the polyphenols found therein). In view of these findings, the present inventors therefore contemplate various compositions, compositions and uses in medical foods, even in medicine, for nutritional supplements and other nutritional products.

Based on his extensive studies, as shown in more detail below, the present inventors have now found that specific mixtures of selected plant materials (and polyphenols contained therein) commonly found in the Mediterranean diet can be prepared which mimic the benefits of the Mediterranean diet, such as with inflammation andmodulation of various biomarkers associated with cellular energy is demonstrated. Among other biomarkers, particularly contemplated biomarkers include various pro-inflammatory cytokines and transcription factors (e.g., TNF- α, IL-6, PGE ₂ Isoprostadin, nfkb), various adipokines (such as leptin, oncostatin M, resistin, FGF21, HGF, and IL-11), mitochondrial biogenesis, cellular glucose uptake, intracellular ATP, antioxidant capacity as measured by reactive oxygen species, and expression of various senescence-associated genes.

Notably, the inventors have found that the compositions according to the inventive subject matter not only inhibit or reduce the production and secretion of pro-inflammatory cytokines that play a role in different inflammatory pathways, but also down-regulate transcription factors critical to the pro-inflammatory response. It is further unexpected that this modulation of inflammatory components is particularly pronounced and synergistically enhanced when a plurality of chemically distinct polyphenol containing plant materials having red, green, orange and violet colors are combined. Even more surprising results, the inventors have found that combining plant materials containing chemically different polyphenols has a much greater benefit than the anti-inflammatory effect. In addition, as shown in more detail below, the compositions of the present invention have significant activity in increasing glucose uptake and mitochondrial biogenesis by cells, increasing ATP levels, and the like. In addition, the composition also exhibits a significant effect on selected fat-releasing hormones associated with inflammation and energy metabolism. Finally, such compositions also reduce gene expression of various senescence-associated genes. These effects, especially in general, appear to indicate the same or similar beneficial molecular mechanisms as are typically observed in the Mediterranean diet. Thus, without wishing to be bound by a particular theory or hypothesis, the inventors believe that the compositions presented herein represent a potentially harmonious combination of biochemically diverse molecules that form the basis of many of the benefits observed with the Mediterranean diet. In this case, it should also be appreciated that various other diets associated with longevity and healthy aging (blue region diets) may be used as sources of the polyphenol components that will collectively enhance pathways and signals critical to health and longevity.

Preferably, the contemplated mixture belongs to a combination of a plurality (e.g., at least two, at least three, or at least four) of colored plant materials of different colors, in particular plant materials having red, green, orange/yellow and/or violet/blue colors. For example, in one embodiment of such a composition, and as shown in more detail below, from red raw materials including apple extract, pomegranate extract, tomato powder, and beetroot powder; a green source material comprising olive extract, rosemary extract, green coffee bean extract and kale; orange/yellow raw materials including onion extract, ginger extract, grapefruit extract, and carrot; purple/blue raw materials including grape extract, blueberry extract, currant powder and elderberry powder to obtain polyphenol-containing products/extracts; specific components and proportions are described in more detail below. Thus, from a different perspective, contemplated compositions will include a substantial amount of polyphenols below at least two, or at least three, or at least four different polyphenols including organic acids, phenols, flavonols, flavanols, anthocyanins, chlorogenic acids, betabetalains, and the like. It will be readily appreciated that the particular choice of plant material will depend on the (polyphenol) component desired in the plant material and its effect on the particular biological system and/or signal pathway.

Of course, it should also be understood that the plant material may be provided in a variety of forms, including all plant material or portions thereof (e.g., roots, fruits, leaves, etc.) in fresh or dried form, juices or macerations of fresh or dried plant material or plant material portions, as well as aqueous or aqueous/alcoholic extracts and chromatographic fractions of such plant material. Still further, it should be noted that one or more polyphenols of the plant material may even be provided as purified (naturally isolated or synthetic) chemical entities, typically having a chemical purity of at least 90%, or at least 95%, or at least 98%, or at least 99%. However, it should be appreciated that in most embodiments, the plant material will be a complex mixture to provide a desired combination of biological effects on a plurality of different molecular entities (e.g., enzymes, receptors, ion channels), wherein at least some biological effects (e.g., at least one, or at least two, or at least three, etc.) are synergistic. Furthermore, it is expected that biological effects on specific molecular entities will also be complementary in biological function, and in some cases even synergistic. Thus, based on the testing and desired targets described in more detail below, it should be appreciated that the compositions of the present subject matter can be formulated to meet specific needs. However, in particularly preferred aspects, contemplated compositions will inhibit multiple targets (e.g., at least two, at least three, at least four, etc.) in multiple and distinct (e.g., at least two, at least three, at least four, etc.) signaling pathways, preferably associated with inflammation, immune response, and/or energy metabolism.

Thus, from a different perspective, it should be appreciated that the mechanism of action of contemplated compositions is not limited to a single specific function (e.g., antioxidants) or to a specific chemical class (e.g., vitamins), but in fact they are complementary and may cooperate to provide multiple biological activities, spanning different metabolic and signaling pathways, and other important cellular health functions such as mitochondrial biogenesis and glucose turnover. Thus, contemplated compositions and methods target a variety of biological systems, including energy metabolism, immune function, inflammation, and the like. From a different perspective, it is contemplated that compositions and methods will reduce inflammation in subjects ingesting such compositions. As used herein, the term "inflammation" refers to a complex series of physiological events including at least one inflammatory component (e.g., an inflammatory disorder, over-expression of pro-inflammatory cytokines, and/or excessive activity of pro-inflammatory signaling pathways) and a decrease in at least one typical age-related cellular energy or mitochondrial biogenesis, and/or sustained or increased expression of senescence-associated genes. In addition, it is contemplated that the plant material will also provide a variety of micronutrients to support or supplement the function of polyphenols and other colored pigments present in the composition.

Among other benefits, it is to be appreciated that the compositions and methods presented herein may be on a variety of pathways that control pro-inflammatory signalingProvides significant complementary activity and therefore will have a more desirable physiological effect profile. For example, as shown in more detail below, contemplated compositions not only exhibit a significant reduction in proinflammatory cytokine release, but also result in nfkb signaling, PGE ₂ Reduction of adipokine and isoprostaglandin release, which are pro-inflammatory mediators that further act in separate signaling pathways. Notably, while the expected sub-mixtures of the compositions do show some effect in modulating the above-described pro-inflammatory mediators, the combination of the sub-mixtures shows a strong synergistic effect in all the above elements. Furthermore, the compositions contemplated herein also exhibit significant antioxidant effects, as determined by the reduction of Reactive Oxidative Species (ROS), which potentially further reduce cellular stress and pro-inflammatory signaling.

It is also notable that contemplated compositions also have a significant impact on cellular energy management, particularly on glucose absorption, mitochondrial biogenesis, and ATP levels. In fact, the contemplated compositions result in improved glucose absorption, which is comparable to, and in some cases even exceeds, that observed for rosiglitazone, a common antidiabetic agent. Advantageously, this increased energy absorption does not result in an increase in free radical production, but contemplated compositions also significantly stimulate mitochondrial biogenesis and an increase in intracellular ATP levels. Thus, the combination of these processes can be seen as counteracting age-related decline in energy metabolism. Still further, regarding age-related decline, the inventors have found that contemplated compositions reduce the expression of many age-related genes.

Thus, it should be appreciated that the compositions contemplated herein may be advantageously used as stand alone products to support various aspects of health and healthy aging, such as support for proper immune function, support for reducing inflammation, and support for glucose levels (especially glucose uptake by cells). From a different perspective, it is to be understood that contemplated compositions may be used to treat or reduce symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress. In this context, it should be noted that the term "support" when used in connection with a physiological function or disorder is intended to prevent a decrease in the activity of one or more components or ingredients associated with the physiological function or disorder, at least partially reverse a decrease in the activity of one or more components or ingredients associated with the physiological function or disorder, maintain the normal function of the activity of one or more components or ingredients associated with the bodily function or disorder, prevent abnormal overactivity (or overexpression) of one or more components associated with the physiological function or disorder, and/or at least partially reverse abnormal overactivity (or overexpression) of one or more components associated with the physiological function or disorder. Alternatively, the compositions described herein may also be combined with other nutritional supplements and/or vitamins to provide benefits not available with such supplements or vitamins.

Regarding the potential molecular mechanisms of the various effects observed using contemplated compositions, the inventors investigated various potential targets that may be upregulated by contemplated compounds, including various cytokines, NF- κb, various fat factors, glucose absorption, mitochondrial biogenesis, ATP levels, reactive oxygen species, expression of senescence-associated genes, and acetylcholinesterase.

Interleukin-6: interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune responses, acute phase responses, and hematopoiesis, and may also play a central role in host defense mechanisms. IL-6 is a pleiotropic cytokine produced by a variety of cells. It acts on a wide range of tissues, and according to the nature of target cells, it exerts growth induction, growth inhibition and differentiation effects, respectively. Notably, IL-6 is not composed of normal cells, but its expression is readily induced by a variety of cytokines, bacterial components such as lipopolysaccharide or viral infections. IL-6 is usually produced at the site of inflammation, and IL-6 binds to its soluble receptor sIL-6Rα, which determines the transition from acute to chronic inflammation by altering the nature of leukocyte infiltration (from polymorphonuclear neutrophils to monocytes/macrophages). In addition, IL-6 has stimulatory effects on T cells and B cells, thereby promoting a chronic inflammatory response. Thus, attempts to down-regulate IL-6 and IL-6 signaling are believed to be effective in treating various chronic inflammatory diseases (e.g., rheumatoid arthritis). As described in more detail below, contemplated compositions have a significant impact on IL-6 release and thus may help to alleviate or prevent one or more symptoms common in (chronic and/or subacute) inflammatory diseases.

Tumor necrosis factor α (TNF- α): tumor necrosis factor-alpha (TNF-alpha), produced mainly by activated macrophages, T lymphocytes and Natural Killer (NK) cells, is a central regulator of inflammation, and TNF-alpha antagonists and inhibitors are interesting approaches to the treatment of inflammatory diseases in which TNF-alpha plays an important pathogenic role. Inhibition of TNF has proven to be an effective treatment for patients suffering from rheumatoid arthritis and other forms of inflammatory diseases, including psoriasis, psoriatic arthritis and ankylosing spondylitis, inflammatory bowel disease. In addition, the efficacy of preventing the infective shock and the AIDS is also provided. Current successful biological therapies include etanercept, infliximab, and the fully human monoclonal antibody adalimumab, among others. However, natural TNF inhibitors such as the compositions described herein provide an attractive route due to the higher cost and side effects of currently known synthetic antibody therapies. Indeed, as described in more detail below, contemplated compositions have a significant inhibitory effect on TNF- α secretion and thus may also help to alleviate or prevent one or more symptoms common in various (chronic and/or subacute) inflammatory diseases.

Prostaglandin E ₂ (PGE ₂ ): prostaglandins (PG) are the major lipid mediators in animals and are synthesized in vivo from arachidonic acid by cyclooxygenase enzymes (COX-1 or COX-2) as rate-limiting enzymes. Prostaglandin E ₂ (PGE ₂ ) Is the most detected PG in a variety of tissues, exerting a variety of physiological and pathological effects through four receptor subtypes (EP 1-4). Non-steroidal anti-inflammatory drugs, such as aspirin and indomethacin, exert potent anti-inflammatory effects by inhibiting COX activity and the resultant inhibition of PG. PGE (PGE) ₂ Regulate acute and chronic inflammation at the molecular and cellular level, as well as autoimmune diseases. PGE (PGE) ₂ In one of acute inflammation, fever and painThe effect is considered to be vasodilation of vascular smooth muscle cells. Subsequent additional data indicate that PGE ₂ Inducing mast cell activation, thereby enhancing vascular permeability, leading to PGE ₂ Induced acute inflammation. In addition, PGE ₂ Promoting Th1 cell differentiation, th17 cell proliferation, and Th22 cell production of IL-22, and exacerbating chronic inflammation and various autoimmune diseases. In addition, PGE ₂ Activation of mast cells via EP3 receptors causes acute inflammation. PGE (PGE) ₂ Chronic inflammation and various autoimmune diseases are also induced by helper T cell 1 (Th 1) differentiation, th17 cell proliferation, and IL-22 production by Th22 cells via EP2 and EP4 receptors. Notably, as shown in more detail below, the inventors have found that the compositions provided herein are useful for PGEs ₂ Has remarkable inhibition effect on synthesis and release. Thus, contemplated compositions will again likely help to reduce or prevent one or more symptoms commonly found in various (chronic and/or subacute) inflammatory and autoimmune diseases, as well as downregulate an exacerbated immune response.

Isoprostadin: isoprostadins are COX-2 independent mediators of the pro-inflammatory process and of enhancing pain perception. The isoprostadins can further cause inflammation by increasing neutrophil adhesion to human venous endothelial cells and by increasing endothelial cell permeability to oxidative stress. Thus, contemplated compositions may also be useful in alleviating or preventing one or more symptoms common in various (chronic and/or subacute) inflammatory diseases, particularly symptoms associated with oxidative stress.

Thus, it should be appreciated that the compositions presented herein have unexpected pleiotropic effects on a variety of different pro-inflammatory signaling elements and signaling pathways, potentially resulting in a broad and systematic reduction of a variety of (chronic and/or subacute) inflammatory conditions. More unexpectedly, the inventors found IL-6, TNF- α and PGE ₂ Is based on the synergistic effect of the various sub-mixtures present in the compositions described herein (see data below), and the sub-mixture inhibition by itself is less pronounced.

In addition to the effects on individual cytokines as described above, the inventors have also found that the compositions provided herein have significant down-regulation effects on NF- κb. In this case, it should be appreciated that NF-. Kappa.B induces the expression of various pro-inflammatory genes, including genes encoding cytokines, chemokines and COX-2, and is involved in the regulation of inflammatory corpuscles. Furthermore, NF- κb plays a key role in regulating survival, activation and differentiation of innate immune cells and inflammatory T cells, and thus has an impact on both innate and target specific immune responses.

NF- κB is highly activated at inflammatory sites of various diseases and can also induce transcription of adhesion molecules, MMPs and Inducible Nitric Oxide (iNOS). For example, in rheumatoid arthritis, NF-. Kappa.B is overexpressed in inflammatory synovial membranes, whose activity can enhance the growth of inflammatory cells and the production of pro-inflammatory mediators such as IL-1, IL-6, IL-8 and TNF-. Alpha.s. Both p50 and p65 localize to the nucleus of synovial lining cells and to monocytes in the sublining region. Helicobacter pylori-associated gastritis also appears to have increased NF- κB activity in gastric epithelial cells, and the number of NF- κB-positive cells correlates with the extent of gastritis. Also, there is evidence that NF-. Kappa.B activation in inflammatory bowel disease, lamina propria macrophages exhibit activated p50, c-Rel, especially p65. Neurological diseases and inflammation associated with atherosclerosis are also mediated to some extent by NF- κb. In addition, human inflammatory airway diseases are also associated with the expression of cytokines and adhesion molecules. This is associated with NF- κB activation in bronchial biopsies of asthmatic patients. An increase in nuclear localized NF-. Kappa.B activity was observed, particularly in airway epithelial cells, with a large number of pro-inflammatory cytokines, chemokines, iNOS and Cox-2 expressed. Other NF- κb-related diseases include atherosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, and systemic inflammatory response syndrome, among others. Thus, compositions that can modulate NF- κb expression may have significant potential in addressing at least some of the symptoms associated with the above-described inflammatory conditions. Thus, contemplated compositions will again likely help alleviate or prevent one or more symptoms common in a variety of (chronic and/or subacute) inflammatory diseases.

Furthermore, particularly in cases where the Body Mass Index (BMI) of the individual is increased or overweight or obese, as discussed and shown in more detail below, contemplated compositions may have a positive effect on adipokine signaling.

The prevalence of obesity worldwide has raised great attention to studies aimed at understanding adipocyte biology and events occurring in adipose tissue and obese individuals. Interestingly, there is increasing evidence that obesity can lead to chronic low (subacute) inflammation, which can lead to systemic metabolic dysfunction associated with obesity-related diseases. In fact, adipose tissue plays a critical endocrine organ role by releasing a variety of bioactive substances, known as fat-derived secreted factors or fat factors, which in some cases have pro-or anti-inflammatory activity. Disorders in the production or secretion of these adipose factors due to dysfunction of adipose tissue may lead to pathogenesis of obesity-related complications.

Proinflammatory adiposity factor: most of the production of adipokines is up-regulated in the obese state and these pro-inflammatory proteins generally act to promote obesity-related metabolic diseases. In addition to leptin, TNF- α and IL-6, recently identified adipokines that promote inflammation include resistin, retinol binding protein 4 (RbP), lipocalin 2, IL-18, angiopoietin-like protein 2 (ANGPTL 2), CC-chemokine ligand 2 (CCL 2), CXC-chemokine ligand 5 (CXCL 5), and nicotinamide ribonucleoside transferase (NAMPT). It is the up-regulation of these factors (and others) that leads to the development of chronic inflammatory states and to metabolic dysfunction.

Anti-inflammatory fat factor: in addition to the numerous pro-inflammatory adipokines described above, adipose tissue also secretes small amounts of anti-inflammatory factors, such as adiponectin, which has been the focus of research, and sFRP5, recently identified as an adipokine.

The inventors have now found that the compositions provided herein completely inhibit the secretion of IGFBP-1, leptin, oncostatin M and resistin TNF- α induced adipocytes with respect to the secretion of adipokines, and the significance of these adipokines is set forth below.

Leptin: the fat factor leptin is the product of the obese gene (ob; also known as Lep), which was identified in ob/ob mice by positional cloning. Leptin regulates feeding behavior through the central nervous system. Leptin deficient mice (ob/ob mice) exhibit hyperphagia (abnormally increased feeding), obesity, and insulin resistance, and administration of leptin to ob/ob mice can reverse these changes. Administration of leptin to fat-bearing mice (lacking subcutaneous adipose tissue and therefore lower leptin levels) can also ameliorate metabolic abnormalities, including insulin resistance and hyperlipidemia. Leptin has also been shown to be effective in ameliorating metabolic dysfunction in lipodystrophy or congenital leptin deficiency patients. However, leptin levels in blood are positively correlated with fat mass, indicating the onset of leptin resistance, and leptin levels in obese subjects are high but without the expected anorexia response. Leptin is similar in structure to the helical cytokine family, including IL-2 and growth hormone 1, and is believed to have pro-inflammatory activity. In fact, leptin increases monocyte production of TNF and IL-6 by activating JAK2 (Janus kinase 2) -sTAT3 (signal transducer and transcriptional activator 3) pathway and stimulates macrophages to produce CC chemokine ligands (i.e., CCL3, CCL4 and CCL 5). Leptin also stimulates RO production in monocytes, promoting cell proliferation and migration reactions. Leptin levels in serum and adipose tissue are elevated in response to pro-inflammatory stimuli, including pro-inflammatory stimuli of TNF and Lipopolysaccharide (LP). In addition, leptin increases production of TH 1-type cytokines IL-2 and IFNγ and inhibits production of TH 2-type cytokine IL-4 by T cells or monocytes, thereby differentiating T cells into a TH1 cell phenotype. Consistent with these findings, leptin deficiency may protect the liver from injury in a T cell mediated hepatitis model. Thus, leptin is widely recognized as a proinflammatory adipokine.

Oncostatin M (OSM): OSM is a gp130 cytokine with its own specific receptor OSMR, heterodimerizes with gp130 and mediates most OSM actions. OSM has substantial sequence identity to Leukemia Inhibitory Factor (LIF) and can regulate various biological processes such as liver development and regeneration, liver insulin resistance and steatosis, inflammation and cardiomyocyte dedifferentiation and remodeling. In addition, OSM contributes to the inflammatory state during obesity and may be involved in the development of insulin resistance.

Resistin: resistin is a member of the cysteine-rich family of resistin-like molecules (RELm) and is involved in the activation of inflammatory processes. Resistin has been shown to induce insulin resistance in mice, and mice lacking resistin have low blood glucose levels after fasting due to low hepatic glucose production. The antibiotic deficiency of ob/ob mice results in increased obesity, but these severely obese mice have improved glucose tolerance and insulin sensitivity. The ability of resistin to regulate glucose metabolism is associated with activation of cytokine signaling inhibitor 3 (sOCs 3) in adipocytes, sOCs3 being an inhibitor of insulin signaling. Although studies in animal models have consistently shown that resistin promotes insulin resistance, the evidence of this effect in humans is unclear. Resistin exists in two quaternary forms: one is a abundant high molecular weight hexamer and the other is a less abundant but more bioactive trimer, which strongly induces liver insulin resistance. In human monocytes, transcription of the resistin gene (RETN) is induced by pro-inflammatory cytokines including IL-1, IL-6 and TNF, whereas in white adipose tissue it is inhibited by the pparγ agonist rosiglitazone, suggesting that the anti-inflammatory effect of rosiglitazone is mediated in part by attenuation of RETN transcription. Recently, studies in mice lacking endogenous resistance expression in adipocytes but expressing human RETN transgenes in macrophages have shown that the pro-inflammatory properties of macrophage derived resistin contribute to insulin resistance in vivo. The pro-inflammatory properties of resistin in human monocytes are apparent because resistin promotes the expression of TNF and IL-6 by these cells. In addition, resistin enhances leukocyte adhesion by promoting expression of pro-inflammatory adhesion molecules vascular cell adhesion molecule 1 (vCAm 1), intercellular adhesion molecule 1 (ICAm 1), and n-pentamin 3 in vascular endothelial cells, directly antagonizing the anti-inflammatory effects of adiponectin on vascular endothelial cells.

IGFBP-1: insulin-like growth factor binding protein 1 (IBP-1), also known as placental protein 12 (PP 12), is a protein encoded by IGFBP1 gene in humans. The protein binds to insulin-like growth factor (IGF) I and insulin-like growth factor (IGF) II and circulates in plasma. Binding of such proteins prolongs the half-life of IGFs and alters their interaction with cell surface receptors. The IGF system is increasingly involved in the development of cardiovascular diseases. The effects of circulating IGF on the vascular system are largely mediated by IGFBP, which controls its entry into cell surface IGF receptors. IGFBP-1 is considered an acute regulator of IGF bioavailability because it is metabolically regulated by sugar regulating hormones. Post-translational phosphorylation of IGFBP-1 significantly increases its affinity for IGF-I, thus representing a further mechanism for controlling IGF bioavailability.

Furthermore, the inventors have found that in relation to the secretion of adipokines, the compositions provided herein stimulate the secretion of ANGPT-L3, C-reactive protein, endocan, FGF-21, HGF, IGFBP-2, IL-11, RBP4 and n-pentamin 2 in TNF- α induced adipocytes (no observable stimulation in TNF- α induced adipocytes alone), and that the significance of these induced adipokines is as follows.

RBP4: serum RbP is a hepatocyte secretion factor responsible for the systemic transport of retinol (vitamin a). Recently, rbP4 was also found to be secreted by adipocytes and macrophages. RbP4 expression is inversely related to the expression of glucose transporter 4 (GluT 4; also known as sLC2A 4) and administration of recombinant RbP4 to normal mice reduces insulin sensitivity. RbP4 is released by adipocytes and inhibits insulin-induced phosphorylation of insulin receptor substrate 1 (IRs 1) in an autocrine or paracrine manner. These data indicate that RbP4 is an adipose tissue-secreting factor, important for regulation of glucose homeostasis in a model of type 2 diabetes.

ANGPTL3: angiopoietin-like protein 3 (ANGPTL 3) is known for its function as a lipoprotein and endothelial lipase inhibitor. Due to the ability of gene or pharmacologic inhibition of ANGPTL3 to significantly reduce circulating lipoproteins, and the cardioprotection of such inhibition, ANGPTL3 has become an emerging therapeutic target, and both antibody and antisense oligonucleotide (ASO) therapies are being tested clinically. Although antibodies are relatively selective for circulating ANGPTL3, ASOs also deplete intracellular proteins and there is new evidence that ANGPTL3 has cellular autonomous function in the liver. These include regulation of hepatocyte glucose and fatty acid absorption, insulin sensitivity, LDL/VLDL residual absorption, VLDL assembly/secretion, polyunsaturated fatty acids (PUFAs) and PUFA-derived lipid mediator content, and gene expression.

Fibroblast growth factor 21: fibroblast growth factor 21 (FGF 21) is a protein encoded by the FGF21 gene in mammals. The protein encoded by the gene is a member of the Fibroblast Growth Factor (FGF) family, particularly members of the endocrine subfamily, including FGF23 and FGF15/19.FGF21 is a major endogenous agonist of the FGF21 receptor and consists of the co-receptors FGF receptor 1 and β -Klotho. FGF21 is a liver factor, a hormone secreted by the liver, signals through FGF21 receptors in the parahypothalamic nucleus, regulates monosaccharide intake and preference for confectionery, and is associated with reduced dopamine transmission in the nucleus accumbens. FGF21 stimulates glucose uptake by adipocytes, but does not work with other cell types. This effect is an addition of insulin activity. Treatment of adipocytes by FGF21 is associated with phosphorylation of FRS2, a protein that links FGF receptors to the Ras/MAP kinase pathway. Injection of FGF21 in ob/ob mice resulted in an increase in Glut1 in adipose tissue. FGF21 can also protect mice from diet-induced obesity and reduce blood glucose and triglyceride levels in diabetic rodents when the transgenic mice overexpress FGF 21. Treatment of mice with FGF21 resulted in increased energy expenditure, fat utilization and lipid excretion.

Interleukin 11: interleukin 11 (IL-11) is an anti-inflammatory cytokine whose receptor is located on most cell types and tissues throughout the body. The anti-inflammatory properties are mediated by inhibition of cytokine synthesis, and in large part by preventing NF- κb activation. Because adipose tissue synthesizes and secretes cytokines that are involved in the establishment of insulin resistance, and because of the ability of IL-11 to inhibit cytokine synthesis, we began a study that established the signaling pathway that IL-11 initiates in adipose tissue.

Hepatocyte Growth Factor (HGF): obesity and its associated chronic inflammation of adipose tissue can trigger insulin resistance, which is associated with a variety of diseases such as hypertension and atherosclerosis. While wild-type mice fed a High Fat Diet (HFD) for 14 weeks had significantly increased body weight and were accompanied by insulin resistance, HGF transgenic mice prevented weight gain and insulin resistance. Accumulation of macrophages and elevation of inflammatory mediator levels in adipose tissue of HGF-transgenic mice were significantly inhibited compared to wild-type mice. HFD-induced obesity showed an increased response to glucose tolerance testing in wild-type mice treated with HGF neutralizing antibodies. These gain-of-function and loss-of-function studies indicate that HFD-induced elevated HGF levels have protective effects on obesity and insulin resistance.

IGFBP-2: proliferation of adipocyte precursors and differentiation thereof into mature adipocytes contribute to the development of obesity in mammals. IGF-I is a potent mitogen and is an important stimulator of adipocyte differentiation. The biological effects of IGF are closely regulated by the IGF-binding protein family (IGFBP), which mainly plays an inhibitory role. IGFBP-2 is the primary binding protein secreted by differentiated white preadipocytes, indicating its potential role in the development of obesity. Studies in transgenic mice that overexpress human IGFBP-2 under the control of their native promoters have shown that IGFBP-2 overexpression is associated with reduced susceptibility to obesity and increased insulin sensitivity. Although wild-type littermates develop glucose intolerance and elevated blood pressure with age, mice overexpressing IGFBP-2 are protected. Furthermore, IGFBP-2 overexpressed mice are resistant to development of obesity and insulin resistance when fed high fat/high energy diet. This lean phenotype is associated with reduced leptin levels, increased glucose sensitivity and reduced blood pressure compared to wild type animals fed a similar high fat diet. These findings indicate that IGFBP-2 plays an important role in obesity prevention.

Glucose uptake and mitochondrial biogenesis: in the elderly, skeletal muscle protein synthesis may resist insulin anabolism. Insulin resistance is also associated with activation of the muscle proteolytic pathway, which can also lead to muscle loss. In turn, muscle is the primary site of insulin-dependent glucose absorption, and insulin-mediated decrease in the surface area of the glucose absorbing muscle can further exacerbate peripheral insulin resistance, resulting in a vicious circle. Oral insulin sensitizers have been reported to maintain muscle mass, but similar associations with muscle strength have not been investigated. Interestingly, skeletal muscle mitochondrial function is reduced in type 2 diabetes and may be improved by peripheral insulin sensitization. Against this background, the inventors have found that the compositions presented herein have a significant beneficial effect on glucose absorption in muscle cells, as described in more detail below. Furthermore, the benefits of the compositions presented herein extend to improvements in mitochondrial function, particularly an increase in mitochondrial biogenesis, as shown in more detail below. Thus, it should be appreciated that the compositions presented herein not only reduce, even reverse, the age-related decline in glucose absorption into muscle, but also increase mitochondrial biogenesis.

Intracellular ATP levels were also tested to determine if the compositions presented herein could increase energy load in cells. As shown below, intracellular ATP levels do improve, particularly at higher concentrations or doses of the intended composition.

Active oxygen: reactive Oxygen Species (ROS) generally include molecular oxygen derivatives that occur as normal byproducts of aerobic metabolism, and increased formation of different ROS often results in molecular damage ("oxidative stress"). ROS, hydrogen peroxide (H) ₂ O ₂ ) And superoxide anion radical (O) ₂ In physiological levels, can act as redox signalling agents, usually under the control of growth factors and cytokines, produced by more than 40 enzymes, mainly including NADPH oxidase and mitochondrial electron transport chains, however, prolonged signalling or ROS levels will lead to oxidative damage and accumulation of metabolic disorders. Thus, the inventors have undertaken to determine whether the intended composition is capable of counteracting oxidative stress. Notably, in addition to all beneficial functions described above, contemplated compositions exhibit significant reduction in ROS, as shown in more detail below.

Furthermore contemplated compositions are based on the composition of the Mediterranean diet, which is known to be common to the longevity population, the inventors have also undertaken to investigate whether the compositions provided herein would have an effect on senescence-associated genes, and the following exemplary results demonstrate significant downregulation of various senescence-associated genes.

Acetylcholinesterase (AChE) is an enzyme that is mainly present in blood and nerve synapses, and plays an important role in various cognitive processes. Acetylcholinesterase catalyzes the hydrolysis of the neurotransmitter acetylcholine to choline and acetic acid, which is the reaction necessary to restore the resting state after cholinergic neuron activation. Acetylcholinesterase inhibitors (e.g., using donepezil) are important targets for the treatment of alzheimer's disease, with acetylcholinesterase inhibitors being the most common drug used to treat alzheimer's disease. In addition to Alzheimer's disease, acetylcholinesterase inhibitors may also be used to diagnose or treat glaucoma, myasthenia gravis, bladder distension, and other disorders. Thus, acetylcholinesterase inhibitors are believed to have a cognitive-promoting effect. To this end, the inventors also determined whether the compositions provided herein have an inhibitory effect on AChE. Notably, as demonstrated in more detail below, the compositions provided herein have significant inhibitory effects on AChE and thus can significantly improve cognition, as well as other benefits.

In a further contemplated aspect of the inventive subject matter, it is to be understood that the compositions provided herein may be formulated in a variety of forms, and particularly preferred formulations include those in combination with a nutritional or pharmaceutically acceptable carrier, most preferably for oral administration (however, parenteral administration is also expressly contemplated). Thus, contemplated compositions may 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, and powders, while other solid formulations include snack bars, fondants, or other edible products, the composition being coated on the edible product (e.g., on a cereal) or the composition being mixed or layered in the other edible product (e.g., into a chewing gum). In 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), functional beverages (e.g., sports or energy beverages), or brewed beverages, or liquid dairy products (e.g., yogurt, kefir).

Thus, contemplated compositions may be provided in bulk as part of an edible or drinkable product, and/or in a single dosage unit for consumption. Most typically, preferably, the contemplated daily dose of the composition (excluding the carrier) is at least 10mg, or at least 50mg, or at least 100mg, or at least 200mg, or at least 300mg, or at least 400mg, or at least 500mg, or at least 750mg, or at least 1000mg, or at least 1500mg. For example, a suitable dosage is 10mg to 50mg, or 50mg to 100mg, or 100mg to 200mg, or 200mg to 400mg, or 300mg to 600mg, or 400mg to 800mg, or 600mg to 1000mg, or 1000mg to 2000mg.

Most typically, contemplated compositions will be administered orally for a prolonged period of time, such as at least 1 week, or at least 2 weeks, or at least 4 weeks, or at least 2 months, or at least 3 months, at least 6 months, or at least 1 year and greater to maintain or support healthy aging. Preferably, the administration is daily, or at least twice a week or at least three times a week. In some embodiments, the contemplated compositions may also be administered topically, typically in the form of a cream, lotion, essence, or elixir or shampoo. In this case, the composition may be a (optionally filtered) aqueous or hydroalcoholic extract, resulting in a clear solution.

As will be readily appreciated, contemplated compositions may also be combined with one or more additional ingredients to impart further desired functionality, and suitable additional ingredients include vitamins (e.g., a single vitamin or vitamin mixture such as a multivitamin mixture), dietary trace elements or minerals (e.g., a single element or mineral, or a mixture of multiple elements or minerals in multiple forms), various specific compounds and mixtures (e.g., prebiotics, human milk oligosaccharides), and/or one or more probiotic microorganisms (e.g., lactobacillus, bifidobacterium, leukotrichia, saccharomyces, etc.), prebiotics (e.g., short chain fatty acids such as butyric acid, secondary bile acids such as (taurine) ursodeoxycholic acid, etc.), dietary fibers (e.g., soluble or insoluble fibers), and/or nutritionally acceptable oligosaccharides (e.g., xylooligosaccharides (XOS) or Fructooligosaccharides (FOS)). Similarly, contemplated compositions may also include niacin, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and/or a nutritionally acceptable CD38 inhibitor, thereby increasing intracellular NAD and/or NADP levels (and their reduced forms NADH and NADPH, respectively). Thus, contemplated compositions may also specifically (synergistically) enhance or support cellular metabolism and energy.

Of course, it should be appreciated that compositions according to the inventive subject matter may be administered not only to humans, but also to other non-human mammals, particularly livestock and companion animals (e.g., dogs, cats, horses). The administration is typically between once a day and three times a day (in some cases even more) over a period of at least two days, three days, five days, one week, 2 to 4 weeks, 1 to 3 months, or even more than 3 months. Most typically, the time of administration is sufficient to at least provide symptomatic relief (e.g., pain and swelling associated with inflammation, low energy levels, frequent infections, etc.), or to prophylactically avoid or help reduce the severity of the health condition.

Examples

Representative compositions:

all experiments were performed using defined mixtures of polyphenol containing products/polyphenol containing extracts common in selected mediterranean diets, unless otherwise indicated. The polyphenol-containing product/polyphenol-containing extract is obtained from a raw material having the following color characteristics: red group (sub-mix): apple extract, pomegranate extract, tomato powder, beetroot; green group (sub-mix): olive extract, rosemary extract, green coffee bean extract and kale; orange/yellow group (sub-mixture): onion extract, ginger extract, grapefruit extract, and carrot; and violet/blue group (sub-mixture): grape extract, blueberry extract, currant powder and elderberry powder. Corn starch, silica and sunflower lecithin are used as processing aids. The relative proportions are shown in table 1 below.

TABLE 1

Phytochemical HPLC/MS analysis: HPLC/MS compositional analysis of the above exemplary compositions reveals the following ingredients and ratios, wherein the columns of each of tables 2-8 indicate 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 3

TABLE 4 Table 4

TABLE 5

TABLE 6

TABLE 7

TABLE 8

The activity of representative compositions was tested for modulation of various target entities associated with mild/chronic inflammation, age-related immune decline, energy metabolism, aging, reactive oxygen species, acetylcholinesterase activity, obesity and viability, unless specifically stated otherwise, with exemplary activity results shown below.

Cytokines:

in the following experiments, the inventionA series of tests were performed by humans to determine the effect of representative compositions and sub-mixtures on various cytokines. For this purpose, the tested compositions were combined with LPS-treated primary human monocytes and SEB-induced T lymphocytes (helper T cells, CD4 ⁺ ) Together to determine a specific response with respect to the selected pro-inflammatory cytokine. Unless otherwise indicated, all test materials were provided as solid powders dissolved in water.

Here, the inventors used primary human monocytes/lymphocytes to test the anti-inflammatory effect of the test article, as described in detail below using the mature and published methods. Human monocytes are one of the major cell types involved in peripheral inflammation, as they are primarily affected by bacterial Lipopolysaccharide (LPS) produced by gram-negative bacteria. In addition, monocyte release of cytokines is also considered to be the first step in the inflammatory cascade, typical cytokines include interleukin-1 (IL-1) beta, IL-6, IL-8, IL-23 and Tumor Necrosis Factor (TNF) alpha, as well as other pro-inflammatory cytokines such as prostaglandin E2 (PGE) ₂ ) Certain chemokines such as monocyte chemotactic protein-1 (MCP-1, also known as CCL 2), and macrophage inflammatory protein-1 (MIP-1) as well as free radical and oxidative stress markers such as isoprostadins. While monocytes can also trigger inflammatory cascades in other cell types, such as fibroblasts and the like, T cells respond to cytokine signals and play a central role in the adaptive immune response.

T cells can be easily distinguished from other lymphocytes by the presence of T Cell Receptors (TCRs) on the surface of the T cells. Interleukin-2 (IL-2) is a 15.5kDa to 16kDa protein consisting essentially of activated CD4 ⁺ T cells (helper T cells) are produced, for example, by enterotoxin type B, also known as Staphylococcal Enterotoxin B (SEB), an enterotoxin produced by the gram positive bacterium staphylococcus aureus. IL-2 regulates the activity of leukocytes (leukocytes, typically lymphocytes) responsible for immunity. IL-2 is part of the natural response of the human body to microbial infection, as well as part of distinguishing between exotic ("non-self") and "self". The parameters studied are recognized mediators of inflammation, all of which are knownIs associated with inflammation and plays an important role in the inflammatory cascade by inducing pain and inflammation.

Determination of primary human monocyte cytokines: human primary monocytes are isolated (enriched) from the buffy coat of a healthy human donor. Cells were seeded in 24-well plates (about 500000 cells/ml in 1 ml) for ELISA experiments. Cells were incubated with LPS (10 ng/ml) for 24 hours. Representative compositions (5 doses) and dexamethasone were added as anti-inflammatory controls 30 minutes prior to LPS treatment (untreated cells as negative control). After 24 hours, the supernatant was removed, centrifuged, and the required inflammatory parameters, such as MCP-1, IL-8, IL-6, IL-23 and TNF-alpha concentrations, were studied using ELISA (MCP-1, TNF-alpha, IL-6, IL-23 and IL-8, R & D/Biotechne) using the manufacturer's protocol. Each dose was studied 6 times in two to three buffy coats from 2 to 3 different donors (n=2 to 3 per buffy coat, n=6 total).

Determination of primary human monocytes PGE2 and isoprostadins (radical markers): human primary monocytes are isolated (enriched) from the buffy coat of a healthy human donor. Cells were seeded in 24-well plates (about 500000 cells/ml in 1 ml) for EIA experiments. Cells were incubated with LPS (10 ng/ml) for 24 hours. Representative compositions (5 doses) and dexamethasone were added as anti-inflammatory controls 30 minutes prior to LPS treatment (untreated cells as negative control). After 24 hours, the supernatant was removed, centrifuged, and the concentration of isoprostadin and PGE2 was studied using EIAs (from Cayman, distributed by Biomol, hamburg, germany). Study was performed 6 times in each dose on two to three buffy coats from 3 different donors (n=2 to 3 per buffy coat, n=6 total).

Determination of IL-2 in primary T cells: human primary T cells are isolated (enriched) from the buffy coat of a healthy human donor. Cells were seeded in 24-well plates (about 500000 cells/ml in 1 ml) for ELISA experiments. Cells were incubated with SEB (1. Mu.g/ml) for 24 hours. Representative compositions (5 doses) and dexamethasone or hydrocortisone were added as anti-inflammatory controls 30 minutes prior to SEB treatment (untreated cells as negative control). After 24 hours, the supernatant was removed, centrifuged, and the IL-2 concentration was studied using ELISA (Biotechne, wiesbaden, germany) using the manufacturer's protocol (other parameters are also possible). Each dose was studied 6 times in two to three buffy coats from 2 to 3 different donors (n=2 to 3 per buffy coat, n=6 total).

As shown in the data shown in fig. 1-7, representative compositions had a significant effect on a variety of pro-inflammatory markers in LPS-treated primary human monocytes. As shown in the summary of fig. 1, representative compositions effectively and significantly inhibited LPS-induced release of IL-6, TNF- α and PGE2 starting from a low dose of 10 μg/ml. LPS-induced release of the isoprostagliptin (PGF 2-alpha), a free radical marker, was inhibited in high doses of the extract. LPS-induced IL-23 release was not affected, whereas IL-8 and MCP1 levels were slightly increased. The composition enhances LPS-mediated release of IL-1β. The anti-inflammatory agent and positive control dexamethasone effectively exclude all parameters except IL-23. Fig. 2 shows the inhibition of various LPS-induced pro-inflammatory cytokines in primary human monocytes by representative compositions (p < 0.05, p < 0.01, and p < 0.001 relative to LPS control (T-test)). More particularly, fig. 3 shows the inhibition of LPS-induced TNF- α release in primary human monocytes (p < 0.05, p < 0.01, and p < 0.001 relative to LPS control (T-test)) by representative compositions. Fig. 4 shows the inhibition of LPS-induced IL-6 release in primary human monocytes (relative to LPS control (T-test), p < 0.05, p < 0.01 and p < 0.001) by representative compositions. Fig. 5 shows the inhibition of LPS-induced PGE2 release in primary human monocytes (relative to LPS control (T-test), p < 0.05, p < 0.01 and p < 0.001) by representative compositions. Fig. 6 shows the inhibition of LPS-induced release of prostaglandins in primary human monocytes (relative to LPS control (T-test), < p < 0.05, < p < 0.01, and < p < 0.001) by representative compositions.

As can be further seen from the data in fig. 7, representative compositions also have a significant effect on IL-2 synthesis in SEB treated primary human monocytes (primary human T cells). Here, representative compositions significantly inhibited LPS-induced IL-2 at higher doses (e.g., 250 μg/ml). Anti-inflammatory and positive control dexamethasone effectively prevented IL-2 synthesis during the course of the experiment as described above. Fig. 7 illustrates the inhibition of SEB-induced IL-2 release in primary human lymphocytes by representative compositions (p < 0.05, p < 0.01, and p < 0.001 relative to SEB control (T-test)). Notably, it should be appreciated that the representative compositions are effective in attenuating or reducing pro-inflammatory signaling while only moderately or not reducing T cell stimulation at all (which is typically required for antigen-specific immune responses).

To investigate any potential active components in a representative composition, the inventors subsequently conducted experiments as shown above, but using only the specific sub-mixtures as described above. To this end, the inventors tested a red group sub-mixture, a green group sub-mixture, an orange/yellow group sub-mixture, and a violet/blue group sub-mixture, and TNF- α, IL-6, and PGE ₂ Typical results for (a) are shown in fig. 8-11 (where p < 0.05, < p < 0.01 and p < 0.001 relative to the LPS control (T-test)). Fig. 8 provides a summary of the green group sub-mixture inhibition data, fig. 9 provides a summary of the red group sub-mixture inhibition data, fig. 10 provides a summary of the orange/yellow group sub-mixture inhibition data, and fig. 11 provides a summary of the violet/blue group sub-mixture inhibition data.

As can be readily seen from the data in fig. 8, inhibition of cytokine release of TNF- α and IL-6 using the green sub-mixture was regulated in a dose dependent manner only, without significant PGE at all concentrations tested ₂ Release inhibition. On the other hand, as shown in fig. 9, no significant inhibition of all cytokines tested was observed when using the red sub-mix. However, IL-6 and TNF- α increased in the higher concentration of the red sub-mixture. Similarly, the orange/yellow sub-mixture did not produce significant inhibition or increase in all cytokines tested as shown in fig. 10. As shown in FIG. 11, there was only a slight inhibition of TNF- α in the case of using the purple/blue sub-mixture.

Notably, it can be quite tolerant from FIG. 12 It is readily seen that (where p < 0.05, p < 0.01 and p < 0.001 relative to the LPS control (T-test)) when four sub-blends were used together, TNF- α, IL-6 and PGE were observed ₂ Is a strong inhibitory synergy of (a). This synergistic profile is not only unexpected, but also strongly suggests that synergistic combinations can be effective in alleviating the signs and symptoms of various inflammatory conditions (e.g., subacute, chronic, disease-related, allergy-related, etc.) in a variety of different pro-inflammatory pathways. This multi-pathway inhibition is considered to be a significantly more effective and physiologically well-tolerated anti-inflammatory immunomodulation than traditional anti-inflammatory drugs. Furthermore, since all of the ingredients in a representative composition are derived from food products, in many cases (due to targeting a variety of different pathways) inflammation can be reduced in a beneficial manner without the side effects common to conventional anti-inflammatory agents.

To investigate whether representative compositions would also have an effect on other tissues (e.g., muscle cells), the inventors determined the mRNA expression of IL-6 and TNF- α in mouse C2C12 muscle cells (immortalized mouse myoblast cell lines). C2C12 cells are supplied by university of koldo, spain. Cells were maintained in Dulbecco's modified Eagle's medium containing 10% Fetal Bovine Serum (FBS), 4.5mM L-glutamine, 1% penicillin and streptomycin antibiotics. For all experiments, cells were grown to 80% to 90% confluency in 24 well plates.

RNA isolation and quantitative PCR: the cultured cells were incubated with representative compositions (1. Mu.g/ml to 100. Mu.g/ml) at various concentrations dissolved in water for 4 hours. Total RNA was extracted using Universal RNA Kit-roboklon. Use of Moloney mouse leukemia Virus (M-MLV) reverse transcriptase (Promega, mannheim, germany), RNase inhibitor(Promega, mannheim, germany), dNTP master mix (Promega, mannheim, germany) and random hexamer primers (Biomers, germany) were reverse transcribed to cDNA from 1. Mu.g of total RNA. Real-time PCR amplification was performed by qTOWER2.0/2.2 quantitative real-time PCR Thermal Cyclers, anaytik Jena using ORA ^TM qPCR Green ROX H Mix,2X-highQu GmbH (g Lai Xida mol)Germany). The reaction conditions were 3 minutes at 95℃followed by 40 cycles of 15 seconds at 95℃30 seconds at 50℃and 45 seconds at 72℃each followed by a plate read. Thereafter, at 95℃for 1 minute, at 55℃for one minute, then at 65℃and 95℃under melt profile conditions, the increment was 0.5℃for 5 seconds, and then the final plate reading was performed. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for sample normalization and data quantification was performed using the comparative cycle threshold Ct method.

The effect of representative compositions on IL-6 and TNF- α mRNA levels in C2C12 muscle cells is depicted in the exemplary results of fig. 13 and 14 (p < 0.05, p < 0.01, p < 0.001 in both results). It is apparent that representative compositions were effective and dose-dependently reducing IL-6 and TNF- α mRNA levels starting at a dose of 10 μg/ml. Thus, it should be appreciated that representative compositions have profound effects on the synthesis and secretion of selected cytokines.

NFκB：

In the following experiments, the inventors performed a series of experiments to determine the effect of representative compositions on nfkb, as NF-kb is one of the most important mediators of pro-inflammatory gene expression. For example, the synthesis of various cytokines such as TNF- α, IL-1β, IL-6 and IL-8 is mediated by NF-. Kappa.B, as is the expression of cyclooxygenase 2 (Cox-2).

To determine NF- κB transcriptional activity, the inventors used an assay kit for HEK293t cells that expressed NF- κB (nuclear factor- κ light chain enhancer of activated B cells) and contained a luciferase reporter gene functionally linked to an upstream NF- κB-genetic response element. Thus, quantifying changes in luciferase expression provides a sensitive surrogate measure of changes in NF- κb activation levels. NF-. Kappa.B is a signal transduction dependent transcription factor. NF- κb reporter cell lines have been shown to provide a strong dose-dependent activation response when treated with tnfα or the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). Thus, the assay is particularly suitable for screening test samples to quantify any functional activity they may induce or inhibit NF- κb activity. Here, NF-. Kappa.B transcriptional activity assays (commercially available from INDIGO Biosciences (Biomol)) were performed according to the manufacturer's instructions.

The effect of representative compositions was observed using the assays described above. It is clear from the data in fig. 15 that at low doses (1 μg/ml and 10 μg/ml, possibly the preparation of the test system) PMA-induced NF- κb activation was enhanced, whereas at high doses, starting from a dose of 50 μg/ml, NF- κb activation was strongly and dose-dependently prevented and almost completely inhibited using extracts of 100 μg/ml and 250 μg/ml, (. P < 0.05, (. P < 0.01, (. P < 0.001)). Notably, this inhibition is more potent than the known NF- κb inhibitor QNZ. As indicated above, this NF- κb inhibition was also consistent with the inhibition observed on various pro-inflammatory cytokines and pathways.

Glucose absorption:

in the following experiments, the inventors performed a series of tests to determine the effect of a representative component on energy metabolism, in particular on glucose absorption.

For this, C2C12 cells (5X 10 ⁴ ) Inoculated in 96-well plates and incubated for 24 hours. The medium was then removed and the cells were cultured in OptiMEM with 50. Mu.M 2-NBDG (2- [ N- (7-nitrobenzene-2-oxa-1, 3-diazol-4-yl) amino)]-2-deoxy-D-glucose labelling and treatment with representative composition or positive control rosiglitazone for 24 hours. The medium was removed, wells carefully washed with PBS and incubated in PBS (100. Mu.l/well). Finally, fluorescence was measured according to the manufacturer's instructions.

Fig. 16 is an exemplary result of the effect of representative components on glucose uptake by mouse C2C12 muscle cells (< p <0.05, < p <0.01, < p < 0.001). As can be easily seen from the data in the figures, the effect of the representative composition on glucose absorption is very high and does have an effect comparable to that of the known antidiabetic compound rosiglitazone. Thus, it should be appreciated that the representative compositions have significant antidiabetic therapeutic effects and significantly promote cellular energy.

Mitochondrial biogenesis:

in anticipation of a significant increase in available energy substrates, the inventors conducted a series of experiments to investigate whether the increased energy metabolites could be effectively used for glycolysis and cellular respiration. Since cellular respiration requires mitochondrial activity, the inventors sought to determine the effect of representative compositions on mitochondrial biogenesis in neuronal cells.

To this end, neuro-2a (N2 a) (ATCC, manassas, va., USA) cells were cultured in DMEM supplemented with 10% FBS, 2mM 1-glutamine and 1% (v/v) penicillin/streptomycin. Maintaining the cells at 37deg.C with 5% CO ₂ Is in a humid atmosphere.

Measurement of mitochondrial biogenesis. N2a cells were seeded in 96-well plates (3.5X10 per well) ³ Cells), 24 hours later, with an increasing concentration of representative composition (5 doses) in each well for 72 hours. Then Mitotracker Green (100nM;Thermo Fisher Scientific,Waltham,MA,USA) was added to the medium for 30 minutes. Cells were washed with PBS and fresh medium was added. Images were taken and fluorescence measured using a cell imaging system, inticutehd (Essen BioScience, inc., herfordshire, uk). Rosiglitazone served as a positive control.

As can be readily seen from the results of fig. 17, representative compositions significantly increased mitochondrial biogenesis. Unexpectedly, mitochondrial biogenesis using this representative composition was superior to the positive control rosiglitazone in this assay.

ATP：

In the following experiments, the inventors performed a series of tests to determine the effect of representative components on intracellular ATP levels. For this, the inventors used RAW264.1 mouse macrophages and C2C12 mouse myocytes (from Uniklinik Freiburg, germany).

The cells were placed in DMEM complete broth (DMEM complete broth) containing 10% fetal bovine serum and 1% antibiotic penicillin/streptomycin, 37 ℃,5% CO ₂ Is maintained in a humid atmosphere. For ATP assays, RAW cells and C2C12 were assayed at 2x10 ⁴ Density of individual cells/wells seeded atIn 96-well plates and in DMEM medium at 37℃in 5% CO ₂ Is incubated overnight in a humid atmosphere. Cell cultures were then stimulated with representative compositions at selected concentrations (5 doses, n=4). By passing through2.0 cell viability/ATP assay to determine ATP in cells.

Notably, as shown in fig. 18, only a low dose of 1 μg/ml of the representative ingredient had a modest increase in ATP levels in macrophages (where p <0.05, < p <0.01, < p < 0.001). Other doses were not effective. In C2C12 cells, the inhibition of ATP levels by representative ingredients was weak but dose dependent (see fig. 19, where p <0.05, < p <0.01, < p < 0.001).

Reactive Oxygen Species (ROS):

in the following experiments, the inventors performed a series of experiments to determine the effect of representative components on the antioxidant capacity of cells by measuring Reactive Oxygen Species (ROS) in the macrophage model system.

For this, the inventors used RAW 264.1 mouse macrophages from germany Uniklinik Freiburg. The cells were placed in DMEM complete broth (DMEM complete broth) containing 10% fetal bovine serum and 1% antibiotic penicillin/streptomycin at 37 ℃ and 5% CO ₂ Is maintained in a humid atmosphere. Cellular responses were studied in the presence of representative compositions and controls (no compound as negative control, trolox and vitamin C as positive control). From the results of FIG. 19, it is clear that the representative ingredients can effectively reduce H starting from a relatively low dose of 1. Mu.g/ml ₂ O ₂ Activation of induced ROS levels. Notably, maximum ROS inhibition was achieved using 50 μg/ml of DC, which was comparable in potency to Trolox C, one of the most potent and most commonly used antioxidants.

Fat factor:

in the following experiments, the inventors conducted a series of experiments to determine the effect of representative ingredients on various fat factors associated with obesity and metabolic diseases. More specifically, the inventors used 3T3-L1 cells (preadipocyte fibroblasts) in a standard cell culture model and used a proteome analysis kit to determine the expression levels of a set of expanded adipokines.

Low passage 3T3-L1 cells (ATCC) were cultured in DMEM containing 25mM glucose, 4mM L-glutamine and 1mM sodium pyruvate, 10% (v/v) fetal bovine serum (abbreviated as DMEM/FBS). Cells were plated in 12-well plates at 37℃with 5% (v/v) CO ₂ Is cultured in a humidified incubator. Two days after confluence, induced differentiation into adipocytes with a stimulator mixture, 2 μg/mL insulin, 0.5mM isobutyl methylxanthine, 0.25 μΜ dexamethasone and 2 μΜ rosiglitazone (all purchased from Sigma) in DMEM/FBS; . Three days later, the differentiation medium was removed, and the cells were cultured in the post-differentiation medium (DMEM/FBS and 2μg.mL ^-1 Insulin) for a further 6 to 9 days, with medium changes every two days or as needed. Differentiation was monitored daily by microscopy.

After differentiation, cells were washed with PBS, incubated in complete DMEM medium, and treated with TNF alpha (50 ng/ml) for 24 hours in the absence and presence of representative compositions (10. Mu.g/ml and 100. Mu.g/ml in water). The supernatant is collected and assayed for detection of adipokine and other related soluble mediators. The fat factor and other obesity-related proteins were detected using a semi-quantitative proteome archiver mouse fat factor array kit (R & DSsystem; minneapolis, MN, USA) according to manufacturer's recommendations. The pixel density on the developed X-ray film was collected with a scanner and analyzed using the ImageJ processing and analysis program (NIH; bethesda, MD, USA).

The following fat factors and soluble media were monitored simultaneously: adiponectin, agRP, ANGPT-L3, C-reactive protein, DPPIV, endocan, fetuin A, FGF acid, FGF-21, HGF, ICAM-1, IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-5, IGFBP-6, IL-10, IL-11, leptin, LIF, lipocalin-2, MCP-1, M-CSF, oncostatin M, n-pentameric protein 2, n-pentameric protein 3, pref-1, RAGE, RANTES, RBP4, resistin, serpin E1, TIMP-1, TNF-alpha, and VEGF.

FIGS. 19-22 depict selected effects of representative compositions on obesity-related protein secretion in differentiated 3T3-L1 cells. Most notably, tnfα induced secretion of IGFBP-1, leptin, oncostatin M, and resistin, and such secretion was completely inhibited by the representative composition, as shown in fig. 21. In contrast, as shown in FIGS. 22 and 23, ANGPT-L3, C-reactive protein, endocan, FGF-21, HGF, IGFBP-2, IL-11, RBP4 and n-pentameric protein 2 were significantly induced by representative compositions of TNFα+ but not only TNFα, indicating that synthesis of these proteins was induced by representative compositions.

Aging:

in the following experiments, the inventors performed a series of experiments to determine the effect of representative components on the expression of selected senescence marker genes in Normal Human Dermal Fibroblasts (NHDF).

NHDF cells were purchased from (ATCC, germany). NHDF cells were cultured in Dulbecco's modified Eagle's medium containing 10% Fetal Bovine Serum (FBS), 4.5mM L-glutamine, 1% penicillin and streptomycin antibiotics. NHDF cells wet 5% CO at 37 ℃ ₂ And (3) growing in the middle. For all experiments, cells were grown to 80% to 90% confluency in 24 well plates. After 24 hours of cell culture, the medium was changed and NHDF cells were kept for 6 days to age.

Real-time fluorescent quantitative PCR (qPCR) detection was performed on aged normal human dermal fibroblasts. The cultured cells were incubated with representative compositions (1. Mu.g/ml to 100. Mu.g/ml) at various concentrations dissolved in water for 4 hours. Total RNA was extracted using Universal RNA Kit-roboklon. Use of Moloney mouse leukemia Virus (M-MLV) reverse transcriptase (Promega, mannheim, germany), RNase inhibitor(Promega, mannheim, germany), dNTP master mix (Promega, mannheim, germany) and random hexamer primers (Biomers, germany) were reverse transcribed to cDNA from 1. Mu.g of total RNA. Real-time PCR amplification was performed by qTOWER2.0/2.2 quantitative real-time PCR Thermal Cyclers, Anaytik Jena uses ORA ^TM qPCR Green ROX H Mix,2X-highQu GmbH (gram Lai Xida mol, germany). Primers were designed to amplify the primers encoding HGF, c-fos, p16 ^INK And p 21. The reaction conditions were 3 minutes at 95℃followed by 40 cycles of 15 seconds at 95℃30 seconds at 50℃and 45 seconds at 72℃each followed by a plate read. Thereafter, at 95℃for 1 minute, at 55℃for one minute, then at 65℃and 95℃under melt profile conditions, the increment was 0.5℃for 5 seconds, and then the final plate reading was performed. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for sample normalization and data quantification was performed using the comparative cycle threshold Ct method.

Notably, it can be seen from FIG. 24 that the representative ingredients strongly inhibited the senescence-associated genes HGF, c-fos, p16 even at very low doses ^INK And expression of p 21.

Acetylcholinesterase (AChE):

in the following experiments, the inventors performed a series of experiments to determine the effect of a representative ingredient on acetylcholine esters as potential targets for neural function.

For this purpose, the inventors used the amplite colorimetric assay (from AATBioquest) according to the manufacturer's instructions. As negative controls, 40 μl NIC (no inhibitor control) assay buffer, 40 μl AHE solution and 40 μl stock solution of diluted donepezil in assay buffer were pipetted into separate wells of a 96-well plate. For negative control, 10. Mu.l of acetylcholinesterase solution (4 mU/10. Mu.l) of NIC and test solution were added to each well. The control sample and the sample with the representative composition were incubated at room temperature with slow shaking for 15 minutes. To each well 50 μl of acetylcholinesterase working solution was added to make the total acetylcholinesterase assay volume 100 μl/well (t=0 minutes). The reaction was incubated at room temperature for 60 min, protected from light (t=60 min). At time points t=0 min and t=60 min, absorbance was measured at 405nm using a PerkinElmer Victor X52030-0050 multimode plate reader of PerkinElmer (Rodgau, germany).

Exemplary test results are shown in fig. 25, illustrating the effect of representative ingredients on acetylcholinesterase activity. Results are expressed in relative units ± SD (×p <0.05, ×p <0.01, ×p < 0.001). It is readily apparent that the representative ingredients have a significant inhibitory effect on acetylcholinesterase at higher concentrations.

Vitality:

in the following experiments, the inventors performed a series of tests to determine the effect of representative ingredients on macrophage viability. Here, RAW 264.1 mouse macrophages were obtained from Uniklinik Freiburg and cultured in supplemented DMEM medium (DMEM complete medium) containing 10% FBS and 1% antibiotic penicillin/streptomycin at 37℃at 5% CO ₂ Is maintained according to standard protocols.

Cytotoxicity assay by plating RAW cells at 2X10 ⁴ Density of individual cells/well was seeded in 96-well plates in DMEM medium at 37 ℃ at 5% CO ₂ Is incubated overnight in a humid atmosphere. Cell culture was then stimulated with representative ingredients at selected concentrations (7 doses, n=4), and cell viability was determined by Alamar Blue staining with NaF as a toxicity control. Example results are shown in fig. 26. As can be seen from the results, only high doses of 500. Mu.g/ml and above have a certain effect on cell viability and cell metabolism, whereas lower concentrations have no statistically significant effect on cell viability and cell metabolism.

Other aspects, considerations, and contemplations suitable for use herein are disclosed in U.S. patent No. 11,065,295, which is incorporated herein by reference.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the present invention are to be understood as being modified in some instances by the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the particular embodiment. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein.

As used herein, the term "administering" a pharmaceutical or nutraceutical composition refers to the direct and indirect administration of a pharmaceutical or nutraceutical, wherein the direct administration of the pharmaceutical or nutraceutical is typically by a health care professional (e.g., doctor, nurse, dietician, etc.), and wherein the indirect administration comprises the step of providing the pharmaceutical or nutraceutical composition to the health care professional or individual in need thereof for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). It should further be noted that the term "predicting" or "predicting" a disorder, a susceptibility to disease progression, or a response to an intended treatment is intended to encompass predicting (but not treating or diagnosing) a behavior of the disorder, the susceptibility, and/or the response, including the rate of progression, the rate of improvement, and/or the duration of the subject's condition.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the invention and does not pose a limitation on the invention claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

As used in the specification herein and throughout the claims that follow, a myriad of words in front of an element may mean more than one unless the context clearly dictates otherwise. Furthermore, as used in the description herein, unless the context clearly dictates otherwise, the meaning of "in … …" includes "in … …" and "on … …". As used herein, unless the context dictates otherwise, the term "coupled to" is intended to include both direct connections (where two elements connected to each other are in contact with each other) and indirect connections (where at least one additional element is located between two elements). Thus, the terms "coupled to" and "coupled to …" are used synonymously.

It will be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner 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 referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. When the specification or claims refer to at least one selected from A, B, C … … and N. The text should be interpreted as requiring only one of the elements, not a plus N, or B plus N, etc.

Claim (modification according to treaty 19)

1. A nutritional composition comprising:

a nutritionally acceptable carrier in combination with a plant material having red, green, orange and violet colors comprising a plurality of chemically different polyphenols;

wherein the red plant material, green plant material, orange plant material, and violet plant material are present in synergistic amounts that reduce the release of proinflammatory cytokines in human leukocytes.

2. The composition of claim 1, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder, and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract, and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder, and/or wherein the violet plant material comprises grape extract, blueberry extract, currant powder, and elderberry powder.

3. The composition of claim 1 or 2, wherein the release of pro-inflammatory cytokines is tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And release of at least one of the isoprostadins.

4. Composition according to claim 1 or 2Wherein the release of the pro-inflammatory cytokine is tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And release of at least two of the isoprostadins.

5. The composition of claim 1 or 2, wherein the release of the pro-inflammatory cytokine is the release of at least three of tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), prostaglandin E2 (PGE 2) and isoprostadins.

6. The composition of claim 1 or 2, wherein the release of pro-inflammatory cytokines is tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And release of isoprostadins.

7. The composition of claim 1, wherein the composition further reduces expression of nfkb.

8. The composition of claim 1, wherein the composition further increases glucose uptake by cells.

9. The composition of claim 1, wherein the composition further increases the biogenesis of mitochondria within a cell.

10. The composition of claim 1, wherein the composition further reduces oxidative damage caused by reactive oxygen species.

11. The composition of claim 1, wherein the composition further reduces the expression of a proinflammatory adipokine.

12. The composition of claim 1, wherein the composition further reduces expression of a senescence-associated gene.

13. The composition of claim 1, wherein the composition is formulated for oral administration.

14. The composition of claim 13, wherein the composition is configured in single dosage units.

15. The composition of claim 14, wherein a single dosage unit comprises 50mg to 1000mg of the composition.

16. The composition of claim 14, wherein the single dosage unit is formulated as a capsule, a fudge, or a powder.

17. The composition of claim 1, further comprising vitamins, dietary trace elements or minerals, probiotics and/or prebiotics.

18. The composition of claim 1, further comprising niacin, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and/or a nutritionally acceptable CD38 inhibitor.

19. The composition of claim 1, wherein the composition is effective to treat or reduce symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress.

20. A method of supporting the health of a subject comprising administering to an individual the composition of claim 1.

21. The method of claim 20, wherein the composition is administered in an amount effective to treat or reduce symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress.

22. The method of claim 20 or 21, wherein the composition is administered for at least 30 days and/or at a daily dose of 50mg to 1000 mg.

23. Use of the composition of claim 1 to support the health of a subject by oral administration of the composition.

24. The use of claim 23, wherein the composition is administered in an amount effective to treat or reduce symptoms associated with inflammatory disorders, metabolic disorders, neurological disorders, cardiovascular disorders, aging, and/or oxidative stress.

25. The use according to claim 24 or 25, wherein the composition is administered for at least 30 days and/or at a daily dose of 50mg to 1000 mg.

26. A method for reducing inflammation in a subject, comprising:

administering to the subject a nutritionally acceptable carrier in combination with plant material having red, green, orange and violet colors comprising a plurality of chemically different polyphenols;

wherein the combination of plant materials synergistically reduces the release of at least one pro-inflammatory cytokine in the human cell and reduces the expression of at least one senescence-associated gene in the human cell.

27. The method of claim 26, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder, and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract, and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder, and/or wherein the violet plant material comprises grape extract, blueberry extract, currant powder, and elderberry powder.

28. The method of claim 26, wherein the red plant material, green plant material, orange plant material, and violet plant material are part of a mediterranean diet.

29. The method of any one of claims 26 to 28, wherein the pro-inflammatory cytokine is selected from tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And isoprostadins.

30. The method of any one of claims 26 to 28, wherein the red, green, orange and violet plant material are present in a synergistic amount that reduces the release of pro-inflammatory cytokines in human cells.

31. The method of any one of claims 26 to 28, wherein the senescence-associated gene is selected from HGF, c-fos, p16 ^INK And p21.

32. The method of claim 26, wherein the reduction in inflammation comprises a reduction in age-related immunity, a reduction in age-related energy metabolism, a reduction in age-related mitochondrial biogenesis, a reduction in at least one symptom associated with inflammation, and/or a reduction in at least one symptom associated with metabolic syndrome.

33. The method of claim 26, wherein administration of the composition further reduces expression of nfkb, increases glucose uptake into cells, increases mitochondrial biogenesis in cells, reduces oxidative damage caused by reactive oxygen species, reduces expression of pro-inflammatory adipokines, and/or increases intracellular ATP.

34. The method of claim 26, wherein administering to the subject comprises orally administering a plant material comprising a plurality of chemically distinct polyphenols.

35. The method of claim 34, wherein the plant material comprising the plurality of chemically distinct polyphenols is administered at a dose of about 50mg to 1000 mg.

36. The method of claim 34, wherein the plant material comprising a plurality of chemically different polyphenols is formulated as a tablet, beverage, or soft candy.

37. The method of claim 26, wherein the combination of plant materials further comprises vitamins, dietary trace elements or minerals, probiotics, and/or prebiotics.

38. The method of claim 26, wherein the combination of plant materials further comprises niacin, nicotinamide riboside, nicotinamide mononucleotide, nicotinamide adenine dinucleotide, and/or a nutritionally acceptable CD38 inhibitor.

39. The method of claim 26, wherein the plant material comprising the plurality of chemically distinct polyphenols is applied at least once daily for a period of at least 30 days.

40. A method for reducing inflammation in a subject, comprising:

Wherein the red plant material comprises apple extract, pomegranate extract, tomato powder and beet root powder;

wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract and kale powder;

wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder;

wherein the purple blue plant material comprises grape extract, blueberry extract, currant powder and elderberry powder; and

wherein the combination of plant materials synergistically reduces at least one pro-inflammatory cytokine, reduces nfκf signaling, and/or reduces at least one pro-inflammatory adipokine in the subject after administration.

41. The method of claim 40, wherein the combination of plant materials is a synergistic combination with respect to a reduction of at least one cytokine.

42. The method of claim 40 or 41, wherein the combination of plant materials reduces at least one of pro-inflammatory cytokine and nfκf signaling in the subject after administration.

43. The method of claim 40 or 41, wherein the combination of plant materials reduces at least one pro-inflammatory cytokine, nfκf signaling, and at least one pro-inflammatory adipokine in the subject after administration.

44. A method for increasing glucose uptake by a cell, comprising:

contacting the cells with a plant material having red, green, orange and violet colors comprising a plurality of chemically different polyphenols;

wherein the combination of plant materials increases glucose uptake by the cells upon contact.

45. The method of claim 44, wherein the increased uptake of glucose by the cell is similar to or greater than the increased uptake of glucose with the thiazolidinedione.

46. A method for increasing ATP levels in a cell, comprising:

wherein the combination of plant material increases ATP levels in the cells after contact.

47. The method of claim 46, wherein the cells are muscle cells.

48. A plant material having red, green, orange and violet colors comprising a plurality of chemically distinct polyphenols for use in alleviating at least one symptom of inflammation in an individual ingesting the plant material.

49. The use according to claim 48, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder, and/or wherein the violet plant material comprises grape extract, blueberry extract, currant powder and elderberry powder.

50. Use of the nutritional composition according to claim 1 for reducing the release of pro-inflammatory cytokines in human leukocytes, reducing nfkb signaling, increasing glucose uptake by cells, increasing mitochondrial biogenesis in cells, reducing oxidative damage caused by reactive oxygen species, reducing the expression of pro-inflammatory adipokines and/or reducing the expression of at least one senescence-associated gene.

Claims

1. A nutritional composition comprising:

4. The composition of claim 1 or 2, wherein the release of pro-inflammatory cytokines is tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And release of at least two of the isoprostadins.

6. The composition of claim 1 or 2, wherein the release of pro-inflammatory cytokines is tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), prostaglandin E ₂ (PGE ₂ ) And the other of the frontRelease of the hormone.

26. A method for reducing inflammation in a subject, comprising:

Wherein the combination of plant materials reduces the release of at least one pro-inflammatory cytokine in the human cell and reduces the expression of at least one senescence-associated gene in the human cell.

34. The method of claim 26, wherein administering to the subject comprises orally administering a plurality of plant materials comprising chemically different polyphenols.

40. A method for reducing inflammation in a subject, comprising:

wherein the combination of plant materials reduces at least one pro-inflammatory cytokine, nfκf signaling, and/or at least one pro-inflammatory adipokine in the subject after administration.

44. A method for increasing glucose uptake by a cell, comprising:

46. The method of claim 44, wherein the step of contacting comprises oral administration.

47. A method for increasing ATP levels in a cell, comprising:

48. The method of claim 47, wherein the cells are muscle cells.

49. A plant material having red, green, orange and violet colors comprising a plurality of chemically distinct polyphenols for use in alleviating at least one symptom of inflammation in an individual ingesting the plant material.

50. The use according to claim 49, wherein the red plant material comprises apple extract, pomegranate extract, tomato powder, and beetroot powder, wherein the green plant material comprises olive extract, rosemary extract, green coffee bean extract, and collard powder, wherein the orange plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder, and/or wherein the violet plant material comprises grape extract, blueberry extract, currant powder, and elderberry powder.

51. Use of the nutritional composition according to claim 1 for reducing the release of pro-inflammatory cytokines in human leukocytes, reducing nfkb signaling, increasing glucose uptake by cells, increasing mitochondrial biogenesis in cells, reducing oxidative damage caused by reactive oxygen species, reducing the expression of pro-inflammatory adipokines and/or reducing the expression of at least one senescence-associated gene.