Compositions and methods for nutritional supplements

Abstract

To provide improved compositions with beneficial / synergistic combination of polyphenols.SOLUTION: The present invention provides compositions for inhibiting one or more enzymes involved in pathways associated with health and healthy aging, the enzymes being selected from a group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3, the composition comprising chemically distinct polyphenols, the polyphenols including organic acids, phenols, flavonols, flavanols, anthocyanins, chlorogenic acids, and betacyanins, the polyphenols being derived from plant material having a red, green, orange-yellow, and purple-blue color, and the polyphenols being in a synergistic combination with respect to inhibition of at least one biochemical marker selected from a group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3.SELECTED DRAWING: Figure 32

Description

Related applications

[0001] This application claims the interests of our concurrently pending U.S. Provisional Patent Application No. 62 / 970,615 filed on 5 February 2020, U.S. Provisional Patent Application No. 63 / 010,183 filed on 15 April 2020, and U.S. Provisional Patent Application No. 63 / 086,207 filed on 1 October 2020, which are incorporated herein by reference in their entirety. [Technical Field]

[0002] The field of the present invention is the composition and methods of nutritional supplements, and in particular the use thereof in conditions, disorders, and diseases related to polyphenols and polyphenol mixtures commonly associated with diets rich in fruits and vegetables, and various enzymes inhibited by such polyphenol mixtures. [Background technology]

[0003] The background information includes information that helps to understand this disclosure. It is not an admission that any of the information provided herein is prior art or relating to the currently claimed invention, nor is it an admission that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications described herein are incorporated by reference to the same extent as any individual publication or patent application is specifically and individually indicated to be incorporated by reference. If a definition or use of a term in an incorporated reference conflicts with or contradicts a definition of a term provided herein, the definition provided herein shall prevail, and the definition of the term in the reference shall not prevail.

[0005] There are a considerable variety of vitamins and other isolated nutritional compounds, and the claimed benefits of such compounds include immune support, anti-inflammatory effects, anti-aging effects, heart support, and digestive support, among many other effects. Unfortunately, there is very little evidence to demonstrate some aspects of these claimed benefits when these vitamins and other isolated nutritional compounds are taken. Similarly, when nutritional supplements are in the form of extracts or powders of parts of plants, various benefits are advertised, but the actual benefits are little to no evidence. Furthermore, isolated nutritional compounds, as with individual plant extracts and concentrates, do not generally reflect a healthy diet.

[0006] In particular, there are specific geographical and ethnographic dietary types associated with overall health, longevity, and / or physical resilience, and such beneficial effects have actually been well documented and demonstrated. For example, the Mediterranean diet is typically associated with reduced cardiovascular risk factors (see, e.g., Nutrients 2018, 10, 379, doi:10.3390 / nu10030379), reduced inflammatory and metabolic biomarkers, a reduced risk of Alzheimer's disease (see, e.g., J Alzheimers Dis. 2010, 22(2), pp. 483-492), and reduced specific inflammatory markers (see, e.g., Nutrients 2018, 10, 62, doi:10.3390 / nu10010062). Polyphenols are one of the common components found in such diets, and various studies have been published on the specific benefits of polyphenols in individual diets (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 (e.g., Annu. Rev. Food Sci. Technol. 2020.11:10.1-10.38). However, because there are many complex and chemically different polyphenols, many studies either focus only on a single polyphenol and the specific biochemical effects of such compounds, or provide general epidemiological information without a more detailed molecular characterization of the diet.

[0007] Various supplements are known to supplement the diet with multiple polyphenols. For example, Vital Reds (manufactured by Gundry MD) offers a concentrated polyphenol powder mixture commercially available from many red plant materials to increase energy and improve digestion. Such mixtures favorably contain a variety of chemically different polyphenols. However, the selection of plant materials used as sources of polyphenols does not reflect typical dietary intake. Similarly, Oxxynea from Fytexia, a commercially available mixture of grape, olive, pomegranate, green tea, grapefruit, bilberry, and orange extracts, is offered as an antioxidant preparation to protect cells from oxidative stress (see, for example, Oxxynea from Fytexia). While beneficial in reducing oxidative stress, the source ingredients of such antioxidant preparations do not reflect typical dietary intake. Surprisingly, despite the many beneficial ingredients contained in various dietary supplements, there are no supplements that are expected to provide the various benefits of the Mediterranean diet, particularly the benefits of the colored polyphenol components of the Mediterranean diet.

[0008] Therefore, despite the existence of various dietary supplements known in the art, all or almost all of them suffer from various drawbacks. Thus, there is a need to provide improved compositions and methods for beneficial / synergistic combinations of polyphenols, which are known to be associated with dietary supplements, particularly healthy diets such as the Mediterranean diet. [Overview of the project]

[0009] The inventors have discovered various compositions and methods for specific combinations of polyphenols and / or polyphenol-rich materials (e.g., extracts and powders) that, when combined, yield many benefits associated with the advantages of the Mediterranean diet. In fact, the compositions and methods disclosed herein have yielded substantial, and in some cases significant, synergistic effects on various molecular biomarkers associated with the benefits of the Mediterranean diet, such as aging, inflammation, immune function, NAD / energy metabolism, and gut microbiota markers.

[0010] In one aspect of the subject matter of the present invention, the inventors intend to create a nutritional composition comprising a nutritionally acceptable carrier in combination with several chemically distinct polyphenols from plant materials having red, green, orange-yellow, and purplish-blue colors. Preferably, the chemically distinct polyphenols from the plant materials exist in a synergistic combination with respect to the inhibition of at least one biochemical marker selected from the group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3.

[0011] For example, in some embodiments, the red plant material comprises at least one (or two, three, or all) of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material comprises at least one (or two, three, or all) of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material comprises at least one (or two, three, or all) of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material comprises at least one (or two, three, or all) of grape extract, blueberry extract, currant powder, and elderberry powder.

[0012] In further aspects of the subject matter of the present invention, chemically distinct polyphenols further inhibit at least one additional biochemical marker selected from the group consisting of ARG-1, ARG-2, SIRT-1, CD39, IDO1, IDO2, NAMPT, PCSK9, and CD47. Furthermore, the intended compositions may further inhibit Keap / Nrf2 binding and / or ACE2 / Spike binding.

[0013] While not mandatory, the composition is most typically formulated as a single oral dose unit (e.g., each containing 50–1,000 mg of the composition), which may be formulated as capsules, gummies, or powder. The composition may also further contain, as needed, vitamins, dietary trace elements or minerals, nicotinamide riboside, probiotics, and / or prebiotics.

[0014] Accordingly, in another aspect of the subject matter of the present invention, the inventors intend to create a nutritional composition comprising a nutritionally acceptable carrier in combination with several chemically different polyphenol-containing plant materials having red, green, orange-yellow, and purplish-blue colors. Most 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-yellow plant material comprises onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material comprises grape extract, blueberry extract, currant powder, and elderberry powder. For example, apple extract, pomegranate extract, olive extract, rosemary extract, green coffee bean extract, onion extract, ginger extract, grapefruit extract, grape extract, and blueberry extract may be ethanol extracts or ethanol / water extracts.

[0015] In such compositions, the combination of plant materials is synergistic with respect to the inhibition of at least one biochemical marker selected from the group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3, and in particular, synergistic with respect to the inhibition of BACE1, CD38, and CD73. More preferred compositions may further inhibit at least one additional biochemical marker selected from the group consisting of ARG-1, ARG-2, SIRT-1, CD39, IDO1, IDO2, NAMPT, PCSK9, CD47, and cathepsin S, and may also inhibit Keap / Nrf2 binding and / or ACE2 / Spike binding.

[0016] As mentioned above, the composition is preferably (but not required) formulated into a single dose unit for oral administration. Typically, the dose unit contains between 50 and 1,000 mg of the composition and is formulated as a capsule, gummy, or powder. If necessary, the composition to be considered may further contain vitamins, dietary trace elements or minerals, nicotinamide riboside, probiotics, and / or prebiotics.

[0017] Among other uses, the intended composition is effective in treating and / or alleviating inflammatory conditions, cardiovascular conditions, neurological conditions, metabolic conditions, and cancer symptoms.

[0018] Accordingly, the inventors also intend to provide methods for supporting an individual's health, comprising the step of administering the compositions presented herein. For example, the compositions may be administered to provide immune support, metabolic support, longevity support, central nervous system (CNS) function support, reduction of inflammatory responses, reduction of the effects of cardiovascular disease, and reduction of amyloid-beta plaque formation rates.

[0019] In further examples of such methods, the composition is administered orally for at least 30 days, typically in a daily dose between 50 and 1,000 mg. As previously mentioned, the composition to be considered may further include the step of co-administering vitamins, dietary trace elements or minerals, nicotinamide riboside, probiotics, and / or prebiotics (this may be done in the same dose unit or separately).

[0020] In another aspect of the subject matter of the present invention, the inventors also intend to reduce the decrease in NAD+ in an individual (e.g., age-related decrease in NAD+), comprising the step of administering to the individual a synergistic combination of polyphenols derived from red, green, orange-yellow, and purple-blue plant materials, such combination synergistically inhibits CD38. In some embodiments, the polyphenols are provided from plant materials.

[0021] Preferably, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder.

[0022] In a further embodiment of the subject matter of the present invention, the inventors have conceived a method to support an individual's longevity, comprising the step of administering to the individual a synergistic combination of polyphenols derived from plant materials having red, green, orange-yellow, and purplish-blue colors, the combination of which synergistically inhibits CD73. In some embodiments, the polyphenols are provided from plant materials.

[0023] Preferably, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beetroot powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, raw coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purple-blue plant material is selected from the group consisting of grape extract, blueberry extract, bilberry powder, and watercress powder.

[0024] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method of supporting an individual's cognitive function, comprising the step of administering a synergistic combination of polyphenols derived from plant materials having red, green, orange-yellow, and purple-blue colors, wherein such a combination synergistically inhibits BACE1. In some embodiments, the polyphenols are provided from plant materials, and the administration increases immune function, thereby maintaining lifespan and / or reducing the rate of amyloid-β plaque formation.

[0025] Preferably, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beetroot powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, raw coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purple-blue plant material is selected from the group consisting of grape extract, blueberry extract, bilberry powder, and watercress powder.

[0026] In yet another aspect of the subject matter of the present invention, the inventors contemplate a method of supporting an individual's central nervous system (CNS) function, comprising the step of administering a synergistic combination of polyphenols derived from plant materials having red, green, orange-yellow, and purple-blue colors, wherein such a combination synergistically inhibits CDK5. In some embodiments, the polyphenols are provided from plant materials, and the administration alleviates age-related decline in cognitive function.

[0027] Preferably, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder.

[0028] In a further aspect of the subject matter of the present invention, the inventors have conceived a method to support immune function in an individual, comprising the step of administering a synergistic combination of polyphenols derived from plant materials having red, green, orange-yellow, and purplish-blue colors, wherein such combination synergistically inhibits at least one of JAK1, JAK2, and JAK3. In some embodiments, the polyphenols are provided from plant materials, and the administration alleviates symptoms of rheumatoid arthritis, psoriasis, or inflammatory bowel disease.

[0029] Preferably, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder.

[0030] Furthermore, the inventors also intend to inhibit at least one of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3, comprising the step of contacting at least one of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3 with a plurality of chemically distinct polyphenols derived from plant material having red, green, orange-yellow, and purplish-blue colors, wherein the chemically distinct polyphenols derived from plant material are synergistic combinations in terms of inhibiting at least one biochemical marker selected from the group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3. In some embodiments, the polyphenols are provided from plant material.

[0031] For example, the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder.

[0032] Advantageously, the contact step is performed in vivo (e.g., oral administration to mammals), and the administration of multiple chemically distinct polyphenols provides immune support, metabolic support, longevity support, central nervous system (CNS) function support, reduces inflammatory responses, mitigates the effects of cardiovascular disease, and / or reduces the rate of amyloid-beta plaque formation.

[0033] From another perspective, the inventors also intend to treat conditions in mammals associated with the activity of at least one of BACE1, CD38, CD73, CDK5, JAK1, JAK2, JAK3, ARG-1, ARG-2, SIRT-1, CD39, IDO1, IDO2, NAMPT, PCSK9, CD47, and cathepsin S. Such methods typically involve administering several chemically distinct polyphenols to a mammal in amounts effective in inhibiting at least one of BACE1, CD38, CD73, CDK5, JAK1, JAK2, JAK3, ARG-1, ARG-2, SIRT-1, CD39, IDO1, IDO2, NAMPT, PCSK9, CD47, and cathepsin S. In preferred embodiments, chemically distinct polyphenols synergistically inhibit BACE1, CD38, CD73, CDK5, JAK1, JAK2, and / or JAK3.

[0034] For example, such conditions may be inflammatory, cardiovascular, neurological, metabolic, and / or cancerous. If necessary, multiple chemically distinct polyphenols may be derived from plant materials having red, green, orange-yellow, and purplish-blue colors. For example, red plant materials may be selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; green plant materials may be selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; orange-yellow plant materials may be selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and purplish-blue plant materials may be selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder. It should also be understood that polyphenols may be provided from plant materials.

[0035] Furthermore, multiple chemically distinct polyphenols are intended to be orally administered to mammals in daily doses typically between 50 and 1,000 mg. If necessary, such methods may further include the step of co-administering to mammals vitamins, dietary trace elements or minerals, nicotinamide riboside, probiotics, and / or prebiotics.

[0036] In further intended embodiments, chemically distinct polyphenols are administered in amounts effective in inhibiting at least three (or at least five, at least ten, or all) of the following: BACE1, CD38, CD73, CDK5, JAK1, JAK2, JAK3, ARG-1, ARG-2, SIRT-1, CD39, IDO1, IDO2, NAMPT, PCSK9, CD47, and cathepsin S.

[0037] Therefore, from another perspective, the inventors also intend to use multiple chemically distinct polyphenols to support healthy aging. Furthermore, it is intended that multiple chemically distinct polyphenols in such use further inhibit SIRT-1, IDO1, IDO2, NAMPT, PCSK9, CD47, Keap / Nrf2 binding, and / or ACE2 / Spike binding, and / or that the chemically distinct polyphenols synergistically inhibit BACE1, CD38, CD73, CDK5, JAK1, JAK2, and / or JAK3.

[0038] As mentioned above, it is preferable that the red plant material is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the green plant material is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the orange-yellow plant material is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the purplish-blue plant material is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder.

[0039] Various objects, features, aspects and advantages of the subject matter of the present invention will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which similar figures represent similar components. [Brief explanation of the drawing]

[0040] [Figure 1] Figure 1 is a graph showing exemplary results of ARG-1 inhibition using the composition relating to the subject of the present invention.

[0041] [Figure 2] Figure 2 is a graph showing exemplary results of ARG-2 inhibition using the composition relating to the subject of the present invention.

[0042] [Figure 3] Figure 3 is a graph showing exemplary results of SIRT1 inhibition using the composition according to the subject of the present invention.

[0043] [Figure 4] Figure 4 is a graph showing exemplary results of Keap1-Nrf2 binding inhibition using the composition according to the subject of the present invention.

[0044] [Figure 5] Figure 5 is a graph showing exemplary results of ACE2-Spike S1 binding inhibition using the composition relating to the subject of the present invention.

[0045] [Figure 6] Figure 6 is a graph showing exemplary results of ACE2-Spike S1 binding inhibition using a multivitamin composition.

[0046] [Figure 7] Figure 7 is a graph showing exemplary results of ACE2-Spike S1 binding inhibition using various further compositions relating to the subject of the present invention.

[0047] [Figure 8]Figure 8 is a graph showing exemplary results of BACE1 inhibition using the composition according to the subject of the present invention.

[0048] [Figure 9] Figure 9 is a graph showing exemplary results of BACE1 inhibition using various further compositions and multivitamin compositions relating to the subject of the present invention.

[0049] [Figure 10] Figure 10 is a graph showing exemplary results of cathepsin S inhibition using the composition according to the subject of the present invention.

[0050] [Figure 11] Figure 11 is a graph showing exemplary results of cathepsin S inhibition using various further compositions relating to the subject of the present invention.

[0051] [Figure 12] Figure 12 is a graph showing exemplary results of cathepsin S inhibition using the composition and multivitamin composition related to the subject of the present invention.

[0052] [Figure 13] Figure 13 is a graph showing exemplary results of CDK5 / p25 binding inhibition using the composition according to the subject of the present invention.

[0053] [Figure 14] Figure 14 is a graph showing exemplary results of CDK5 / p25 binding inhibition using various further compositions and multivitamin compositions relating to the subject of the present invention.

[0054] [Figure 15] Figure 15 is a graph showing exemplary results of IDO1 inhibition using the composition relating to the subject of the present invention.

[0055] [Figure 16]Figure 16 is a graph showing exemplary results of IDO2 inhibition using the composition according to the subject of the present invention.

[0056] [Figure 17] Figure 17 is a graph showing exemplary results of NAMPT inhibition using the composition relating to the subject of the present invention.

[0057] [Figure 18] Figure 18 is a graph showing exemplary results of PCSK9:LDLR binding inhibition using the composition relating to the subject of the present invention.

[0058] [Figure 19] Figure 19 is a graph showing exemplary results of CD47 inhibition using the composition relating to the subject of the present invention.

[0059] [Figure 20] Figure 20 is a graph showing exemplary results of CD38 inhibition using the composition relating to the subject of the present invention.

[0060] [Figure 21] Figure 21 is a graph showing exemplary results of CD38 inhibition using further compositions relating to the subject of the present invention.

[0061] [Figure 22] Figure 22 is a graph showing exemplary results of CD38 inhibition using known compositions containing nicotinamide riboside.

[0062] [Figure 23] Figure 23 is a graph showing exemplary results of CD38 inhibition using the composition relating to the subject of the present invention and known multivitamin compositions.

[0063] [Figure 24] Figure 24 is a graph showing exemplary results of JAK1 inhibition using the composition relating to the subject of the present invention.

[0064] [Figure 25] Figure 25 is a graph showing exemplary results of JAK2 inhibition using the composition relating to the subject of the present invention.

[0065] [Figure 26] Figure 26 is a graph showing exemplary results of JAK3 inhibition using the composition relating to the subject of the present invention.

[0066] [Figure 27] Figure 27 is a graph showing exemplary results of CD39 inhibition using the composition relating to the subject of the present invention.

[0067] [Figure 28] Figure 28 is a graph showing exemplary results of CD39 inhibition using a low-concentration composition related to the subject of the present invention.

[0068] [Figure 29] Figure 29 is a graph showing exemplary results of CD39 inhibition using further compositions relating to the subject of the present invention.

[0069] [Figure 30] Figure 30 is a graph showing exemplary results of CD39 inhibition using a selected composition relating to the subject of the present invention.

[0070] [Figure 31] Figure 31 is a graph showing exemplary results of CD39 inhibition using the composition relating to the subject of the present invention and known multivitamin compositions.

[0071] [Figure 32] Figure 32 is a graph showing exemplary results of CD73 inhibition using the composition relating to the subject of the present invention.

[0072] [Figure 33] Figure 33 is a graph showing exemplary results of CD73 inhibition using a low-concentration composition related to the subject of the present invention.

[0073] [Figure 34] Figure 34 is a graph showing exemplary results of CD73 inhibition using further compositions relating to the subject of the present invention.

[0074] [Figure 35] Figure 35 is a graph showing exemplary results of CD73 inhibition using the composition relating to the subject of the present invention and known multivitamin compositions. [Modes for carrying out the invention]

[0075] The inventors have discovered that specific combinations of polyphenol-containing materials (and the polyphenols found therein) strongly modulate numerous biomarkers associated with the beneficial effects of the Mediterranean diet. Therefore, in light of these findings, the inventors intend to develop various compositions for dietary supplements and other nutritional products, as well as for use in medicinal foods and even in medical applications.

[0076] Most notably, the compositions presented herein exhibit substantial and synergistic effects on numerous biomarkers associated with proper immune and CNS function, effective cellular metabolism, and longevity, and have shown even more significant effects on additional biomarkers associated with inflammatory responses, adverse cardiovascular disease, and / or amyloid-beta plaque formation. For example, the compositions and methods presented herein have demonstrated significant and beneficial effects on multiple enzyme targets involved in various aspects of health and healthy aging, including ARG-1, ARG-2, SIRT1, BACE1, cathepsin S, CDK5, IDO1, IDO2, NAMPT, PCSK9, CD47, CD38, JAK1, JAK2, JAK3, CD39, CD73, and Keap / Nrf2. From a different perspective, it should be understood that the compositions presented herein are useful in beneficially influencing multiple pathways associated with health and healthy aging through the inhibition of key signaling components and / or enzymes in these pathways. Surprisingly, the modulation of these biomarkers observed using the compositions presented herein paralleled the biomarker profiles in individuals adhering to the Mediterranean diet and those with significant longevity.

[0077] For example, arginase 1 (ARG1) and arginase 2 (ARG2) are key enzymes in the urea cycle, cleaving L-arginine to form urea and L-ornithine. While the urea cycle protects against excess ammonia, L-ornithine is necessary for cell proliferation, collagen formation, and other important physiological functions. In particular, increased arginase activity in mammals is associated with cardiovascular, renal, and central nervous system dysfunction and pathology, as well as immune system dysfunction and cancer development. Two important aspects of excessive arginase activity may be involved in disease. First, overactive arginase may reduce the supply of L-arginine necessary for nitric oxide (NO) production by NO synthase. Second, excessive amounts of L-ornithine may cause structural problems in the vascular system, neuronal toxicity, and abnormal proliferation of tumor cells (see, e.g., Physiol Rev 98:641-665, 2018). Furthermore, studies have demonstrated that increased reactive oxygen species formation and key inflammatory mediators promote pathological elevation of arginase activity. Therefore, inhibition of ARG1 and ARG2 is considered beneficial in counteracting the adverse effects of arginase hyperactivity. As detailed below, the proposed composition was effective in inhibiting ARG1 and ARG2 activity even at relatively low concentrations.

[0078] In another example, sirtuin 1 (SIRT1) is a nuclear enzyme that deacetylates transcription factors contributing to cellular regulation, particularly the response to stressors. For instance, SIRT1 deacetylates members of the PGC1-α / ERR-α complex, an important metabolic regulatory transcription factor, and deacetylates / inactivates the p53 protein, a key factor in many neoplastic diseases. SIRT1 has also been shown to play a role in activating T helper 17 cells, which contribute to autoimmune diseases. As will be shown in more detail below, the proposed composition was effective in mildly inhibiting SIRT1 activity at relatively moderate concentrations.

[0079] In yet another example, β-secretase 1 (BACE1) has been shown to be essential for β-amyloid production in Alzheimer's disease and has been reported to be associated with cognitive decline and central nervous system (CNS) function decline (see, e.g., Molecules. October 13, 2017, 22(10), p. 1723). Indeed, β-amyloid accumulation is a characteristic of aging, and such inhibitory compounds are thought to beneficially slow or halt β-amyloid accumulation, thereby maintaining or preserving cognitive ability and CNS function. As will be described in more detail below, the compositions considered have shown remarkable, potent, and even synergistic effects with respect to BACE1 inhibition.

[0080] In another example, it has been reported that higher levels of cathepsin S are associated with a higher risk of death, and some studies have also reported an increased risk of cardiovascular death (e.g., JAMA, September 14, 2011, Vol. 306, No. 10, pp. 1113). Other experimental studies have suggested that cathepsin S activity is involved in the development of cardiovascular disease through the promotion of atherosclerotic plaques and the destabilization of advanced plaques. Furthermore, cathepsin S activity is also involved in cancer development through the stimulation of cancer cell migration and tumor angiogenesis, and higher levels of cathepsin S activity have been reported to correlate with brain aging. Therefore, a decrease in cathepsin S activity levels is thought to beneficially counteract these risks. In particular, the intended composition showed a remarkable and potent inhibitory effect with respect to BACE1 inhibition.

[0081] Similarly, cyclin-dependent kinase 5 (CDK5) is known to be essential for proper CNS function, and its role in peripheral tissues and diseases is increasing. For example, acute CDK5 inhibition has high potential therapeutic value to prevent nerve damage in cases of stroke or brain injury, or during high-risk surgeries such as neurovascular or cardiovascular surgery, or potentially during complex, long-term childbirth. Pharmacological inhibition of CDK5 has been shown to protect neurons under various stressful conditions and aging (see Curr Pharm Des. 2016, 22(5), pp. 527-534, etc.). Here again, the intended composition showed a remarkable, potent, and synergistic inhibitory effect with respect to CDK5 inhibition.

[0082] In yet another example, Janus kinases 1, 2, and 3 (JAK1, JAK2, and JAK3) are involved in the regulation of the cell cycle and cancer, possibly via NF-κB activation (see, e.g., Cells 2020, 9, pp. 1451). Furthermore, small molecule drugs that inhibit Janus kinases (JAK1-3) are important signaling mediators downstream of many pro-inflammatory cytokines and are attracting attention as a safe and effective option for immune-mediated diseases. Unsurprisingly, JAK inhibition has emerged as a major therapeutic option for inflammation-related conditions such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and cardiovascular disease. In particular, the intended composition showed remarkable and potent synergistic inhibitory effects (especially at high concentrations) with respect to JAK1, JAK2, and JAK3 inhibition.

[0083] In yet another example, inhibition of indoleamine 2,3-dioxygenase-1 (IDO1) has emerged as a novel therapeutic strategy for immune support, attracting particular attention in the restoration of tumor-mediated immunosuppression (see, e.g., Cancer Res. 2017, December 15, 77(24), pp. 6795-6811). Similarly, indoleamine 2,3-dioxygenase-2 (IDO2) has recently been implicated in cancer, inflammation, and immune regulation, and considerable resources have been devoted to identifying IDO2-specific inhibitors. Increased IDO1 and IDO2 activity has also been observed as a function of aging (e.g., Immunity & Aging, 2011, 8:9), with age-related increases in inflammation, autoimmune diseases, and malignancies. Surprisingly, as will be shown in more detail below, the compositions of the present invention exhibited potent inhibitory effects with respect to IDO1 and IDO2 inhibition.

[0084] Regarding energy metabolism, NAMPT and CD38 are known to regulate intracellular NAD+ and are considered essential for maintaining and supporting cellular energy and proper metabolic function. In fact, CD38 hyperactivity has been reported to increase with aging (see, e.g., Biochem Biophys Res Commun. May 28, 2019, 513(2), pp. 486-493), cancer and age-related diseases (see, e.g., Trends Pharmacol Sci. April 2018, 39(4), pp. 424-436), and immunomodulatory and metabolic diseases (see FIMMU, May 2019, Vol. 10, p. 1187, etc.). Against this backdrop, it is thought that inhibiting CD38 may mitigate or even prevent diseases associated with CD38 hyperactivity. On the other hand, nicotinamide phosphoribosyltransferase (NAMPT) mediates the rate-limiting step of the NAD salvage pathway, which maintains cellular bioenergy and provides substrates necessary for essential functions in cells, especially rapidly proliferating cancer cells. Here again, the intended composition showed potent and synergistic inhibitory effects with respect to CD38 inhibition and potent inhibition of NAMPT activity.

[0085] Furthermore, hyperactivity of CD39 and CD73 has been reported to contribute to immunosuppression, suppression of checkpoint inhibition in tumors, and other aspects of immunomodulation (see, e.g., FIMMU, June 2017, Vol. 8, p. 727; Immunol Rev. March 2017, 276(1), pp. 121-144). In addition, studies have shown that individuals over 100 years of age have significantly lower CD39 and CD73 expression compared to younger individuals (in this case, those in their 80s), suggesting that CD39 and CD73 mRNA levels may be characteristic of human aging. Alternatively, since aging is associated with a decline in immune function, individuals become more susceptible to infections and have a higher incidence of malignant diseases. Therefore, a decrease in CD39 activity is thought to be directly related to healthy aging and longevity. Surprisingly, the intended composition had a significant and synergistic effect in inhibiting CD73 and a remarkable inhibitory effect on CD39 activity, as will be shown in more detail below. Similarly, CD47 inhibition has been shown to lead to stimulation of phagocytosis in cancer cells, and CD47 blockade is associated not only with enhancing the function of innate immune cells but also with adaptive immune responses (see, for example, Cell Reports 31, 107494, April 14, 2020). In particular, the proposed composition had a significant inhibitory effect on CD47, as will be further detailed below.

[0086] In yet another example, the proprotein convertase subtilisin / kexin type 9 (PCSK9) has been reported to be a contributing factor to plasma cholesterol levels, and PCSK9-targeting inhibitors have been shown to lower human plasma cholesterol. Here again, as will be shown in more detail below, the intended composition had a significant synergistic effect in inhibiting PCSK9.

[0087] Furthermore, the Keap1-Nrf2 pathway is a major regulator of cytoprotective responses to endogenous and exogenous stress caused by reactive oxygen species (ROS), and Nrf2 activates the expression of various genes encoding stress response proteins. Therefore, inhibitors of Keap1-Nrf2 binding are thought to increase the expression of stress response-related proteins. Surprisingly, as will be shown in more detail below, the intended composition had a significant effect in inhibiting Keap1-Nrf2 binding (and thus increasing the availability of Nrf2).

[0088] Finally, the inventors also discovered that the intended composition has an inhibitory effect on ACE2-Spike protein binding, which is involved in the proliferation of coronaviruses, particularly SARS-CoV-2. Thus, as can be easily understood, the intended composition may provide at least some protective effect against coronaviruses, particularly SARS-CoV-2.

[0089] Based on extensive research, the inventors have found that it is possible to prepare specific blends of selected plant materials common in the Mediterranean diet that mimic the benefits of the Mediterranean diet, as demonstrated by the adjustment of biomarkers. Preferably, such blends are combinations of colored plant materials belonging to a number of different color groups (e.g., at least two, at least three, or at least four), and in particular, plant materials having red, green, orange-yellow, and / or purplish-blue colors. For example, in one embodiment of the composition to be considered, the polyphenol-containing product / extract is obtained from red raw materials including apple extract, pomegranate extract, tomato powder, and beet root; green raw materials including olive extract, rosemary extract, green coffee bean extract, and kale; orange-yellow raw materials including onion extract, ginger extract, grapefruit extract, and carrot; and purplish-blue raw materials including grape extract, blueberry extract, currant, and elderberry, the specific components and proportions of which are described in more detail below. Therefore, from a different perspective, the intended composition contains numerous polyphenols of at least two, at least three, or at least four different polyphenol classes, including organic acids, phenols, flavonols, flavanols, anthocyanins, chlorogenic acid, betacyanins, etc. As is readily apparent, the specific selection of plant material depends on the desired (polyphenol) components in the plant material and their effects on specific biological systems and / or signaling pathways.

[0090] Of course, it should be understood that plant materials can be provided in a variety of forms, including whole plant materials or parts thereof in fresh or dried form (e.g., roots, fruits, leaves, etc.), juices or macerates from fresh or dried plant materials or parts thereof, and aqueous or aqueous / alcoholic extracts and chromatographic fractions of the aforementioned plant materials. Furthermore, it should be noted that one or more polyphenols of the plant material may be provided as purified (naturally isolated or synthetic) chemicals having a chemical purity of typically at least 90%, or at least 95%, or at least 98%, or at least 99%. However, in most embodiments, plant materials are complex mixtures that provide a combination of desired biological effects on a number of different molecular entities (e.g., enzymes, receptors, ion channels), and at least some of these biological effects (e.g., at least one, or at least two, or at least three, etc.) are synergistic. Furthermore, the biological effects on specific molecular entities are also considered complementary in their biological function. Therefore, it should be understood that, based on the tests and desired objectives described in more detail below, the subject matter compositions of the present invention can be formulated to satisfy specific needs. However, in a particularly preferred embodiment, the intended composition inhibits multiple targets (e.g., at least two, at least three, at least four, etc.) in multiple distinct (e.g., at least two, at least three, at least four, etc.) signaling pathways.

[0091] Therefore, from another perspective, the mechanism of action of the intended compositions is not limited to a single specific function (e.g., antioxidant) or a specific chemical category (e.g., vitamin), but rather provides multiple biological activities across different metabolic and signaling pathways in a complementary and synergistic manner. Thus, the intended compositions and methods target various biological systems, including energy metabolism, immune function, nerve and CNS function, cardiac function, and inflammation. In particular, as described in more detail below, the compositions intended herein have also influenced many biomarkers associated with longevity (e.g., in blue zone regions such as the Mediterranean region and the Okinawa region). Furthermore, plant materials are thought to also provide various micronutrients to support or complement the functions of polyphenols and other colored pigments present in the compositions.

[0092] Therefore, it should be understood that the compositions contemplated herein can be advantageously used as standalone products to support various aspects of health and healthy aging, such as supporting proper immune function, supporting inflammation reduction, supporting normal NAD+ levels, and supporting heart health. In this context, it should be noted that the term “support,” when used in relation to a physiological function or condition, is intended to mean preventing a decrease in the activity of one or more components or components related to a physiological function or condition, at least partially reversing a decrease in the activity of one or more components or components related to a physiological function or condition, maintaining the normal function or activity of one or more components related to a physiological function or condition, preventing abnormal hyperactivity (or overexpression) of one or more components related to a physiological function or condition, and / or at least partially reversing abnormal hyperactivity (or overexpression) of one or more components related to a physiological function or condition. Alternatively, the compositions contemplated herein can also be combined with other dietary supplements and / or vitamins to provide beneficial effects that cannot be obtained otherwise from such supplements or vitamins. In this regard, it should be understood that, as will be shown in more detail below, most, if not all, of the biomarkers tested herein were not substantially modulated by the multivitamin compositions. Therefore, it should be recognized that the compositions presented herein offer a new and distinct class of health support, with various beneficial effects beyond those of multivitamin preparations.

[0093] In a further intended embodiment of the subject matter of the present invention, the compositions presented herein may be formulated in a variety of forms, particularly preferred formulations including those combined with nutritionally or pharmaceutically acceptable carriers, and it should be understood that they are most preferably for oral administration (however, parenteral administration is also explicitly intended). Thus, the compositions to be formulated may be formulated as solid or liquid products. For example, if the compositions to be formulated as solid products, suitable product forms include single-dose unit formulations such as capsules, tablets, and powders, while other solid formulations include snack bars, gummies, or other edible products to which the compositions are coated (e.g., on cereal), or the compositions are mixed or layered (e.g., on chewing gum). In another example, if the compositions to be formulated as liquid products, suitable product forms include flavored and / or carbonated beverages (e.g., tea, juice), functional beverages (e.g., sports or energy drinks) or intravenous fluids, or liquid dairy products (e.g., yogurt, kefir).

[0094] Therefore, the intended composition may be provided in large quantities as part of an edible or beverage product, and / or in single dose units for consumption. Most typically, the daily dose of the intended composition (excluding the carrier) is preferably at least 10 mg, or at least 100 mg, or at least 200 mg, or at least 300 mg, or at least 400 mg, or at least 500 mg, or at least 750 mg, or at least 1,000 mg, or at least 1,500 mg. For example, suitable doses are between 10 and 100 mg, or between 100 and 200 mg, or between 200 and 400 mg, or between 300 and 600 mg, or between 400 and 800 mg, or between 600 and 1,000 mg, or between 1,000 and 2,000 mg.

[0095] As is easily understood, the intended composition may be combined with one or more additional components to impart further desired functionality, and suitable additional components include vitamins (e.g., single vitamins or vitamin blends such as multivitamin blends), dietary trace elements or minerals (e.g., individual elements or minerals, or mixtures of multiple elements or minerals in various forms), various specialty compounds and mixtures (e.g., compositions containing nicotinamide riboside, prebiotics, breast milk oligosaccharides), and / or one or more probiotic microorganisms (e.g., Lactobacillus species, Bifidobacterium species, Leuconostoc species, Saccharomyces boudii, etc.).

[0096] Of course, it should be recognized that the compositions relating to the subject matter of the present invention can be administered not only to humans but also to other non-human mammals, particularly livestock and companion animals (e.g., dogs, cats, and horses). Administration is typically carried out once to three times a day (or more) for a period of at least two, three, five days, one week, two to four weeks, one to three months, or even longer. Most typically, administration is carried out for a period of time sufficient to provide at least symptom relief of the condition (e.g., pain and swelling associated with inflammation, low energy levels, frequent infections, etc.) or to preventively avoid or reduce the severity of the health condition. Examples Typical compositions:

[0097] Unless otherwise specified, all tests were performed using a defined mixture of selected polyphenol-containing products / extracts common to the Mediterranean diet. The polyphenol-containing products / extracts were derived from raw materials characterized by color, as follows: Red group: apple extract, pomegranate extract, tomato powder, and beetroot; Green group: olive extract, rosemary extract, green coffee bean extract, and kale; Orange-yellow group: onion extract, ginger extract, grapefruit extract, and carrot; Purple-blue group: grape extract, blueberry extract, currant, and elderberry. Corn starch, silica, and sunflower lecithin were used as processing aids. Relative proportions are shown in Table 1 below. JPEG0007884042000001.jpg128166

[0098] Phytochemical HPLC / MS / MS Analysis: HPLC / MS compositional analysis of the above exemplary composition revealed the following components and proportions. Here, each column in Tables 2-8 shows the sample ID (col.1), chemical substance (col.2), MH (col.3), RT (col.4), peak intensity (col.5), and MS / MS fragment (col.6). JPEG0007884042000002.jpg89166JPEG0007884042000003.jpg216166JPEG0007884042000004.jpg113166JPEG000 7884042000005.jpg215166JPEG0007884042000006.jpg215166JPEG0007884042000007.jpg26166JPEG0007884042 000008.jpg216166JPEG0007884042000009.jpg53166JPEG0007884042000010.jpg216166JPEG0007884042000011. jpg220166JPEG0007884042000012.jpg73166JPEG0007884042000013.jpg33166JPEG0007884042000014.jpg62166

[0099] The biological activity of the compositions was tested for inhibition of various target entities central to various pathways associated with health and healthy aging, and exemplary activity results are shown below. More specifically, the inventors tested the compositions for inhibition of human ARG-1, ARG-2, SIRT1, BACE1, cathepsin S, CDK5, IDO1, IDO2, NAMPT, PCSK9, CD47, CD38, JAK1, JAK2, JAK3, CD39, and CD73, as well as for Keap / Nrf2 binding inhibition and ACE2-Spike binding inhibition. ARG1 and ARG2:

[0100] In the following experiments, the inventors sought to determine whether representative compositions affect ARG1 and ARG2. The reagents used are shown in Tables 9-10 below, and were tested as described unless otherwise noted (nor-NOHA is the reference compound). JPEG0007884042000015.jpg31166JPEG0007884042000016.jpg33166

[0101] Assay conditions: Analysis was performed using human recombinant ARG1 or ARG2 as the enzyme and thioarginine as the substrate. UV absorbance at 412 nm correlated with the amount of ARG1 / ARG2 reaction product. Test samples (HP Color Blend) were dissolved in 1% (w / v) 70% (w / v) ethanol (EtOH) and filtered through 0.22 μm. Nor-NOHA was also dissolved in 10 mM 100% (w / v) DMSO. 5 μl of 20X sample solution was added to 90 μl of substrate, and the reaction was started by adding 5 μl of 20X ARG1 / ARG2 solution. For the negative control (blank), 5 μl of assay buffer was added instead of ARG1 / ARG2. The resulting 100 μl reaction mixture contained the indicated amount of sample, 225 μM thioarginine, detection reagent, and 30 nM ARG1 or 5 nM ARG2 in 1X ARG assay buffer. All reactions were carried out at room temperature and incubated for 30 minutes to eliminate solvent influence, ensuring that all wells containing either the sample or control contained a final concentration of 0.7% (v / v) ethanol. UV absorbance was read at 0 and 30 minutes, and net values ​​were obtained. Absorbance was measured using a Tecan Infinite M1000 microplate reader.

[0102] Data Analysis: The experiment was performed twice at each concentration. The data was analyzed using the software GraphPad Prism. When the compound was not present, the net absorbance (At) for each dataset was defined as 100% activity. When ARG1 / ARG2 was not present, the net absorbance (Ab) for each dataset was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = [(AA b ) / (A t -A b )] × 100, where A is the absorbance in the presence of the compound. Percent inhibition was calculated according to the following formula: % inhibition = 100 - % activity.

[0103] Results: In particular, significant inhibitory activity was found for both ARG-1 and ARG-2 across all test concentrations, as shown in Tables 11-12 below. As is readily apparent from the results, the inhibition was significant compared to the reference inhibitor and, as can be seen from Table 13, was not specific to either ARG-1 or ARG-2. The results for ARG-1 and ARG-2 are also shown in Figures 1 and 2, respectively. JPEG0007884042000017.jpg61166JPEG0007884042000018.jpg61166JPEG0007884042000019.jpg51166 SIRT1:

[0104] In the following experiments, the inventors sought to determine whether representative compositions affect SIRT1. The reagents used are shown in Tables 14-15 below, and unless otherwise specified, the tests were performed as described (suramin was used as the reference compound). JPEG0007884042000020.jpg51166JPEG0007884042000021.jpg28166

[0105] Assay conditions: Samples were dissolved in 70% ethanol. Serial dilutions of the compounds were performed first in 70% ethanol, with a maximum concentration of 1%. Each intermediate compound dilution (in 70% ethanol) was then directly diluted 10-fold with the assay buffer for intermediate dilutions in 7% ethanol in the HDAC assay buffer. 5 μl of the dilution was added to 50 μl of the reaction mixture, so that the final ethanol concentration for all reactions was 0.7%.

[0106] The enzymatic reaction of the SIRT1 enzyme was carried out twice at 37°C for 30 minutes using a 50 μl mixture containing SIRT assay buffer, 5 μg of BSA, HDAC substrate (see 2.3.1), SIRT enzyme, and the test compound. After the enzymatic reaction, 50 μl of 2x SIRT Developer was added to each well for the SIRT enzyme, and the plate was incubated at room temperature for a further 15 minutes. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader at 360 nm excitation and 460 nm emission.

[0107] Data Analysis: SIRT activity assays were performed twice for each concentration. Fluorescence intensity data were analyzed using the computer software Graphpad Prism. If the compound was not present, the fluorescence intensity (F) of each dataset was calculated. t ) was defined as 100% activity. If SIRT is not present, the fluorescence intensity (F) of each dataset was defined. b ) was defined as 0% activity. The percentage activity of each compound in the presence of FF was calculated according to the following formula: % activity = (FF b ) / (F t -F b ), where F is the fluorescence intensity in the presence of the compound.

[0108] Results: The inhibition results are shown in the table below. The percentage inhibition of each compound against SIRT1 is summarized. The reference compound, test composition, and HP color blend precipitated at the 0.01% intermediate dilution stage. Figure 3 shows the results in graph format, and Table 16 provides the results in numerical format. For easy understanding, the tested compositions exhibited significant SIRT1 inhibitory activity. JPEG0007884042000022.jpg80166 Keap / Nrf2:

[0109] In a further series of experiments, the inventors investigated whether the test composition could interfere with the Keap1-Nrf2 bond, and exemplary results are provided below. The reagents used are shown in Table 17 below and were tested as described unless otherwise specified (the reference compound was LDEETGEFL-OH). JPEG0007884042000023.jpg48166

[0110] Assay conditions: The test compound was diluted with 7% ethanol, and 5 μl of the dilution was added to 50 μl of the reaction solution to make the final concentration of ethanol 0.7%. The reference compound was diluted with 10% DMSO, and 5 μl of the dilution was added to 50 μl of the reaction solution so that the final concentration of DMSO was 1%. The binding reaction was carried out at room temperature for 30 minutes in a 50 μl mixture containing 10 mM HEPES, pH 7.4, 50 mM EDTA, 150 mM NaCl, 0.05% Tween 20, 0.01% BSA, 100 nM Keap1, 5 nM fluorescent probe, and the test compound. Fluorescence intensity was measured at an excitation of 475 nm and an emission of 520 nm using a Tecan Infinite M1000 microplate reader.

[0111] Data analysis: All binding assays were performed twice in 96-well plates. Tecan Magellan6 software was used to convert the fluorescence intensity to fluorescence anisotropy. The fluorescence anisotropy data were analyzed using computer software, Graphpad Prism. The fluorescence anisotropy (F At ) of the samples containing KeapI and the probe in each data set was defined as 100% activity. The fluorescence anisotropy (F Ab ) of the samples containing the compound but not KeapI in each data set was defined as 0% activity. The percent binding efficacy in the presence of the competing compound was calculated according to the following formula. JPEG0007884042000024.jpg16113

[0112] Here, FA represents the fluorescence anisotropy in the presence of the compound. Then, the % binding values were plotted as a bar graph as shown in Figure 4, and the numerical results are shown in Table 18 below. JPEG0007884042000025.jpg48166

[0113] As can be easily seen from the results in the table and Figure 4 above, the tested compositions exhibited considerable inhibitory activity against Keap1-Nfr2 binding. ACE2-Spikes:

[0114] In this series of experiments, the inventors investigated whether possible compositions and fractions could reduce the binding of the SARS-CoV-2 spike protein to ACE2, and whether vitamins also had any effect.

[0115] The reagents used are shown in Tables 19-21 below, and unless otherwise specified, the tests were performed as described (* indicates a reference compound). In this series of experiments, the representative compositions described above were compared to their individual colors as shown below, and also to multivitamin preparations as shown below. Table 19 shows the representative compositions, and Table 20 shows the subfractions of the colors of the representative compositions, with DC-5 = yellow blend, DC-9 = purple blend, DC-21 = green blend, and DC-13 = red blend. Here, as described above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. Table 21 shows the multivitamin blends, and Table 22 shows the ACE2 / Spike reagents used. JPEG0007884042000026.jpg41166JPEG0007884042000027.jpg55166JPEG0007884042000028.jpg36166JPEG0007884042000029.jpg25166

[0116] Assay conditions: Nickel plates were coated with 50 μl of 1 μg / ml ACE2-His at room temperature for 1 hour, washed, and blocked before initiating the reaction. 10 μl of compound solution was incubated in ACE2-His coated assay wells with 20 μl of 1X immunobuffer for 30 minutes, after which 20 μl of 5 μg / ml Spike S1-Spike was added to initiate the reaction. A control of the same concentration as the solvent (ethanol) was included in the study. The reaction was allowed to proceed at room temperature for 1 hour. Next, the wells were washed three times with 1X immunobuffer and blocked with blocking buffer 2 for 10 minutes. 100 μl of streptavidin-HRP was added to all wells and incubated for 1 hour. Finally, the plate was emptied, washed three times, blocked, and then 100 μl of freshly prepared HRP chemiluminescent substrate was added to all wells. Immediately thereafter, the luminescence of the samples was measured using a BioTek Synergy 2 microplate reader.

[0117] Data Analysis: Luminescence data was analyzed and compared. In the absence of a compound, the intensity of each dataset (C) was measured. e ) was defined as 100% activity. In the absence of the enzyme, the intensity (C0) of each dataset was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (C e -C0), where C is the luminescence in the presence of the compound.

[0118] The results for representative compositions are shown in Table 23 and Figure 5, and the results for multivitamin preparations are shown in Table 24 and Figure 6. The results for colored blends are shown in Table 25 and Figure 7. As can be easily seen from the data in Tables 23-25 ​​and Figures 5-7, representative compositions showed synergistic inhibition of ACE2-spike S1 binding, especially at low concentrations, while multivitamin preparations did not show a significant inhibitory effect. Furthermore, the selected DC-13 and DC-9 also had some inhibitory effect on their own. JPEG0007884042000030.jpg92166JPEG0007884042000031.jpg75166JPEG0007884042000032.jpg133166 BACE1:

[0119] In a further series of experiments, the inventors attempted to determine whether representative compositions and their fractions, as well as multivitamin mixtures, affect the activity of recombinant human BACE1 using in vitro fluorescence assays.

[0120] The reagents used are shown in Tables 26-28 below, and the tests were performed as described unless otherwise specified (*verbecestat was used as the reference compound). Table 26 shows typical compositions, and Table 27 shows the colored fractions and multivitamin mixes. Here, DC-5 = yellow blend, DC-9 = purple blend, DC-21 = green blend, DC-13 = red blend DCH-TIV 1.0 (Centrum Vitamin for Adults). As mentioned above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. JPEG0007884042000033.jpg33166JPEG0007884042000034.jpg57166JPEG0007884042000035.jpg28166

[0121] Assay conditions: 80 μl of BACE1 was incubated with 10 μl of sample and reference compound for 10 minutes. The reaction was then initiated by adding 10 μl of BACE1 FRET peptide substrate, and the product kinetics were measured for 1 hour using an Infinite M1000 microplate reader (Tecan). 80 μl of assay buffer was added to the blank control well instead of the enzyme. All wells contained ethanol at a final assay concentration of 0.7% (v / v).

[0122] Data Analysis: All conditions were repeated twice for each concentration. Fluorescence intensity data was analyzed using Prism (GraphPad). When the compound was absent (control), the fluorescence intensity (Ft) in each dataset was defined as 100% activity. When the enzyme was absent (blank control), the fluorescence intensity (Fb) in each dataset was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (F - Fb) / (Ft - Fb), where F is the fluorescence intensity in the presence of the compound. The net signal, measured in relative fluorescence units (RLU), was obtained by subtracting the initial fluorescence.

[0123] As can be seen from the data in Table 29 and Figure 8, representative compositions exhibited substantial inhibitory activity against the tested BACE1. Surprisingly, as can be seen from the data in Table 30 and Figure 9, the colored fraction also provided significant inhibition of BACE1. Furthermore, these data clearly showed that the BACE1 inhibition was synergistic, while the tested multivitamins had virtually no inhibitory effect. JPEG0007884042000036.jpg59166JPEG0007884042000037.jpg109166 Cathepsin S:

[0124] In yet another series of experiments, the inventors attempted to determine, using in vitro enzyme assays, whether representative compositions and their fractions, as well as multivitamin mixtures, affect the activity of recombinant human cathepsin S.

[0125] The reagents used are shown in Tables 31-33 below, and the tests were performed as described unless otherwise specified (*E-64 was used as the reference compound). Here, the names and components of D5, D9, D13, and D21 are as described above. JPEG0007884042000038.jpg36166JPEG0007884042000039.jpg49166JPEG0007884042000040.jpg38166

[0126] Assay conditions: Cathepsin S was activated by diluting concentrated stock with acidic assay buffer at room temperature for 30 minutes. Next, 5 μl of sample or reference inhibitor was added to 20 μl of enzyme solution and pre-incubated for 30 minutes. The enzymatic reaction was initiated by adding 25 μl of fluorescent substrate, bringing the total reaction volume to 50 μl. The reaction time was 60 minutes, after which fluorescence intensity at 360 nm excitation and 460 nm emission was read using a Tecan Infinite M1000 microplate reader.

[0127] Data Analysis: Enzyme activity assays were performed twice at each concentration. Fluorescence intensity data were analyzed and compared. In the absence of the compound, the intensity of each dataset was defined as 100% (Ce) activity. In the absence of the enzyme, the intensity of each dataset was defined as 0% (C0) activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (Ce - C0), where C is the intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table). The fluorescence of the compound was removed by subtracting the fluorescence at reaction time = 0.

[0128] The results of the above tests are shown in Tables 34-36. Table 34 and Figure 10 show the results for representative compositions, Table 35 and Figure 11 show the results for various colored fractions, and Table 36 and Figure 12 show the results for the multivitamin mix (DCH-TIC-0.5 is the representative composition, and DCH-TIC-1.0 is the centrum multivitamin mix). As can be easily seen from the results, the representative compositions and colored blends D9 and D13 significantly inhibited cathepsin S, but the multivitamin mixture had no substantial effect. JPEG0007884042000041.jpg67166JPEG0007884042000042.jpg105166JPEG0007884042000043.jpg69166 CDK5:

[0129] In a further series of experiments, the inventors attempted to determine whether representative compositions and their fractions, as well as multivitamin mixtures, affect the enzyme activity of recombinant human CDK5 / p25 using in vitro enzyme assays.

[0130] The reagents used are shown in Tables 37-39 below, and unless otherwise specified, the tests were performed as described (Dinaciclib was used as the reference compound). Here, the names and components of D5, D9, D13, and D21 are as described above. JPEG0007884042000044.jpg38166JPEG0007884042000045.jpg96166JPEG0007884042000046.jpg31166

[0131] Assay conditions: Analysis was performed using the Kinase-Glo Plus luminescence kinase assay kit (Promega). Kinase activity was measured by quantifying the amount of ATP remaining in the solution after the kinase reaction. The luminescence signal from the assay correlated with the amount of ATP present and inversely correlated with the amount of kinase activity. The reference compound was diluted in 10% DMSO, and 5 μl of the dilution was added to 50 μl of the reaction mixture so that the final concentration of DMSO was 1% in all reactions. The test compound was diluted with water, and 5 μl of the dilution was added to 50 μl of the reaction mixture. All enzymatic reactions were carried out at 30°C for 45 minutes. The 50 μl reaction mixture contained 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg / ml BSA, 1 mM DTT, 10 μM ATP, kinase substrate, and enzyme. After the enzymatic reaction, 50 μl of Kinase-Glo Plus luminescence kinase assay solution (Promega) was added to each reaction mixture and incubated on a plate at room temperature for 15 minutes. The luminescence signal was measured using a BioTek Synergy 2 microplate reader.

[0132] Data Analysis: Kinase activity assays were performed twice at each concentration. Luminescence data were analyzed using the computer software Graphpad Prism. 100% activity (Lut-Luc) was defined as the difference in luminescence intensity between the absence of kinase (Lut) and the presence of kinase (Luc). Using the luminescence signal in the presence of the compound (Lu), % activity was calculated as follows: % activity = {(Lut-Lu) / (Lut-Luc)} × 100%, where Lu is the luminescence intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table).

[0133] The results of the above tests are shown in Tables 40-42, with Table 40 and Figure 13 showing the results for representative compositions, and Table 41 and Figure 14 showing the results for various colored fractions and a multivitamin mixture (referred to here as DCH-TIV 1.0). As can be easily seen from the presented data, the representative composition and its fraction showed a significant inhibitory effect on CDK5, while the multivitamin mix showed virtually no significant inhibitory effect compared to the representative composition. Furthermore, at least at medium and low concentrations, CDK5 inhibition showed at least a moderate synergistic effect in the representative composition. JPEG0007884042000047.jpg63166JPEG0007884042000048.jpg121166 IDO1 / IDO2:

[0134] In a further series of experiments, the inventors attempted to determine, using UV absorbance assays, whether representative compositions affect the enzyme activity of recombinant human IDO1 and / or IDO2.

[0135] The reagents used are shown in Tables 42-43 below, and unless otherwise specified, the tests were performed as described. JPEG0007884042000049.jpg41166JPEG0007884042000050.jpg26166

[0136] Assay Conditions: The assay was performed by measuring UV absorbance using recombinant IDO and L-tryptophan as substrates. UV absorbance at 321 nm correlates with the amount of N-formylkynurenine, the reaction product of IDO. The compound (see 2.2) was diluted with 20% DMSO, and 10 μl of the dilution was added to 200 μl of the reaction mixture to ensure a final DMSO concentration of 1% in all reactions. All reactions were carried out at room temperature. 200 μl of the reaction mixture in the IDO assay buffer contained 400 nM IDO1 or IDO2, the indicated amounts of the inhibitor, tryptophan, and the coupled reaction components. The incubated reaction mixture was incubated for 180 minutes before reading the UV absorbance. For the negative control (blank), 10 μl of the assay buffer was added instead of the IDO enzyme. Absorbance was measured using a Tecan Infinite M1000 plate reader.

[0137] Data Analysis: The experiment was performed twice at each concentration. Data was analyzed using the computer software GraphPad Prism. When the compound was not present, the absorbance signal (At) in each dataset was defined as 100% activity. When IDO was not present, the absorbance signal (Ab) in each dataset was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = [(A - Ab) / (At - Ab)] × 100, where A is the absorption signal in the presence of the compound. The percentage inhibition was calculated according to the following formula: % inhibition = 100 - % activity.

[0138] The results of the above tests are shown in Tables 44-45. Table 44 and Figure 15 show the results for representative compositions regarding IDO1 inhibition, while Table 45 and Figure 16 show the results for representative compositions regarding IDO2 inhibition. As can be easily understood from the presented data, the representative compositions showed inhibitory effects on IDO1 at high concentrations and significant inhibitory effects on IDO2 at high and medium concentrations. JPEG0007884042000051.jpg65166JPEG0007884042000052.jpg61166 NAMPT:

[0139] In yet another series of experiments, the inventors attempted to determine whether representative compositions affect the enzyme activity of recombinant human NAMPT using in vitro enzyme assays.

[0140] The reagents used are shown in Tables 46-47 below, and unless otherwise specified, the tests were performed as described. JPEG0007884042000053.jpg43166JPEG0007884042000054.jpg30166

[0141] Assay conditions: The control compound was dissolved in DMSO. The compound was first diluted with 100% DMSO, with a maximum concentration of 0.01 mM. Next, each intermediate compound dilution (in 100% DMSO) was directly diluted 10-fold with the assay buffer to obtain intermediate dilutions of 10% DMSO in the assay buffer. The final concentration of DMSO was 1% in the reaction of the control compound and NAMPT only. In the case of NAMPT, the compound (see 2.2) was pre-incubated with the NAMPT enzyme (see 2.3.1) for 30 minutes. All enzymatic reactions were carried out twice at 30°C for 120 minutes by adding a substrate mixture contained in a 50 μl mixture of 50 mM Tris-HCl, pH 8.0, 12.5 mM MgCl2, 20 μM nicotinamide, 40 μM PRPP, 20 μM ATP, 30 μg / mL alcohol dehydrogenase, 10 μg / mL NMNAT, 1.5% alcohol, 1 mM DTT, 0.02% BSA, and 0.01% Tween. The final concentration of DMSO in all reactions was 1%. Fluorescence intensity was measured using a Tecan Infinite M1000 microplate reader with excitation at 340 nm and emission at 460 nm.

[0142] Data Analysis: The NAMPT activity assay was performed twice at each concentration. Fluorescence intensity data were analyzed using the computer software GraphPad Prism. When the compound was absent, the fluorescence intensity (Ft) in each dataset was defined as 100% activity. When NAMPT was absent, the fluorescence intensity (Fb) in each dataset was defined as 0% activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (F - Fb) / (Ft - Fb), where F is the fluorescence intensity in the presence of the compound. The percentage activity values ​​were plotted on a bar graph.

[0143] The results of the above experiment are shown in Table 48 and Figure 17. As can be easily understood, representative compositions had an inhibitory effect on NAMPT. JPEG0007884042000055.jpg67166 PCSK9:

[0144] In further experiments, the inventors attempted to determine whether representative compositions affect the binding of recombinant human PCSK9 and LDLR using in vitro ELISA.

[0145] The reagents used are shown in Tables 49-50 below, and unless otherwise noted, the tests were performed as described (anti-PCSK9 was used as the reference compound). JPEG0007884042000056.jpg38166JPEG0007884042000057.jpg26166

[0146] Assay conditions: Before initiating the reaction by adding 20 μl of 2.5 ng / μl PCSK9-biotin, 5 μl of sample or reference inhibitor was pre-incubated in the assay wells with 25 μl of 1X PCSK9 assay buffer. The reaction was then allowed to proceed at room temperature for 2 hours. Next, the wells were washed three times with 1X PCSK9 assay buffer and blocked with blocking buffer for 10 minutes. 100 μl of streptavidin-HRP was added to all wells and incubated for 1 hour. Finally, the plate was emptied, washed three times, blocked, and then 100 μl of freshly prepared HRP chemiluminescent substrate was added to all wells. Immediately thereafter, the luminescence of the samples was measured using a BioTek Synergy 2 microplate reader.

[0147] Data Analysis: Binding activity assays were performed twice at each concentration. Emission signals were analyzed and compared. In the absence of the compound, the signal in each dataset was defined as 100% (Ce) activity. In the absence of the ligand (no LDLR), the signal in each dataset was defined as 0% (C0) activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (Ce - C0), where C is the intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table). Fluorescence of the compound was removed by subtracting the fluorescence at reaction time = 0.

[0148] The results of the PCSK9 binding inhibition assay are shown in Table 51 and Figure 18. As is clear from the data, representative compositions showed significant inhibitory activity against the binding of recombinant human PCSK9 to LDLR. JPEG0007884042000058.jpg65166 CD47:

[0149] In further experiments, the inventors also attempted to determine, using in vitro binding assays, whether representative compositions affect the binding activity of recombinant human CD47 (hCD47) to human SIRP-α (hSIRP-α).

[0150] The reagents used are shown in Tables 52-53 below, and unless otherwise noted, the tests were performed as described (anti-PCSK9 was used as the reference compound). JPEG0007884042000059.jpg37166JPEG0007884042000060.jpg40166

[0151] Assay conditions: 50 μL of CD47 was used at a concentration of 2 ng / μL and coated overnight at 4°C. After washing and blocking steps, the test compound was added to the CD47 coated plate, followed by SIRP-α-biotin. The reaction was incubated at room temperature for 2 hours. Binding was detected using HRP-conjugated streptavidin.

[0152] Data Analysis: Binding assays were performed twice at each concentration. Luminescence data were analyzed using the computer software GraphPad Prism. Percent inhibition was determined by normalizing the data against signals from negative control wells (uncoated wells treated with biotinylated ligand, set as 100% inhibition) and positive control wells (coated wells treated with biotinylated ligand in the absence of the inhibitor, set as 0% inhibition).

[0153] The results of the above experiment are listed in Table 54 and Figure 19. To reiterate, it can be seen that the representative compositions had a significant inhibitory effect on the binding activity between recombinant human CD47 (hCD47) and human SIRP-α. JPEG0007884042000061.jpg85166 CD38:

[0154] In a separate series of experiments, the inventors attempted to determine whether representative compositions and their fractions, as well as multivitamin mixtures and other compounds known to affect NAD+ levels, affect the hydrolase activity of recombinant human CD38 using in vitro enzyme assays.

[0155] The reagents used are shown in Tables 55-59 below, and the tests were performed as described unless otherwise specified (*apigenin was used as the reference compound). Table 55 shows a typical composition, and Table 56 shows the colored fractions and multivitamin mixtures. Here, DC-5 = yellow blend, DC-9 = purple blend, DC-21 = green blend, DC-13 = red blend DCH-TIV 1.0 (Centrum Multivitamin for Adults). Here, as described above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. Table 57 shows two compounds known to affect NAD+ levels. Both commercially available "Elysium Health NAD" and "TrueNiagen" contain nicotinamide riboside, and Table 58 shows representative compositions (DCH-TIV-0.5) and a multivitamin composition (DCH-TIV-1.0 (Centrum Multivitamin for Adults)). Table 59 shows the enzymes and substrates used in this series of experiments. JPEG0007884042000062.jpg36166JPEG0007884042000063.jpg43166JPEG0007884042 000064.jpg49166JPEG0007884042000065.jpg42166JPEG0007884042000066.jpg27166

[0156] Assay conditions: 10 μl of sample or reference inhibitor was added to 20 μl of enzyme solution and pre-incubated for 30 minutes. The enzymatic reaction was initiated by adding 20 μl of substrate ε-NAD+, with a total reaction volume of 50 μl. The reaction time was 10 minutes, after which fluorescence intensity at 300 nm excitation and 410 nm emission was read using a Tecan Infinite M1000 microplate reader.

[0157] Data Analysis: Enzyme activity assays were performed twice at each concentration. Fluorescence intensity data were analyzed and compared. When the compound was absent, the intensity of each dataset was defined as 100% (Ce) activity. When the enzyme was absent, the intensity of each dataset was defined as 0% (C0) activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (Ce - C0), where C is the intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table). Fluorescence of the compound was removed by subtracting the fluorescence at reaction time = 0.

[0158] The results for the above substrates are shown in Tables 60-63 and Figures 20-23. More specifically, Table 60 and Figure 20 show the results when representative compositions were used to inhibit CD38. As can be easily seen from the data, the representative compositions showed remarkably strong inhibition. Table 61 and Figure 21 show the results when the colored fractions of the representative compositions were used to inhibit CD38. In particular, strong inhibition was observed here as well. Furthermore, it should be noted that the colored fractions in the representative compositions produced a strong synergistic effect with respect to CD38 inhibition.

[0159] Table 62 and Figure 22 show the results of corresponding experiments in which “Elysium Health NAD” and “TrueNiagen” (both containing nicotinamide riboside) were used for CD38 inhibition. Here, both formulations showed CD38 inhibition, but not to the same extent as the representative composition. In contrast, Table 63 and Figure 23 show the results of corresponding experiments in which a multivitamin composition was used for CD38 inhibition. Here, a direct comparison is shown between the representative composition (DCH-TIV-0.5) and the multivitamin composition (DCH-TIV-1.0 in Table 63, DCH-TIG-1.0 in Figure 23). JPEG0007884042000067.jpg64166JPEG0007884042000068.jpg111166JPEG0007884042000069.jpg81166JPEG0007884042000070.jpg81166 JAK1 / JAK2 / JAK3:

[0160] In further experiments, the inventors attempted to determine, using in vitro enzyme assays, whether representative compositions and their fractions affect the enzymatic activity of recombinant human kinases JAK1, JAK2, and JAK3.

[0161] The reagents used are shown in Tables 64-66 below, and the tests were performed as described unless otherwise specified (*apigenin was used as the reference compound). Table 64 shows the typical composition, and Table 65 shows the colored fractions. Here, DC-5 = yellow blend, DC-9 = purple blend, DC-21 = green blend, and DC-13 = red blend DCH-TIV 1.0 (Centrum Multivitamin for Adults). As mentioned above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. Staurosporine was also used as the reference compound. Table 66 shows the enzymes and substrates used in the assay. JPEG0007884042000071.jpg47166JPEG0007884042000072.jpg83166JPEG0007884042000073.jpg50166

[0162] Assay conditions: Analysis was performed using the Kinase-Glo Plus Luminescent Kinase Assay Kit (Promega). Kinase activity was measured by quantifying the amount of ATP remaining in the solution after the kinase reaction. The luminescence signal from the assay correlates with the amount of ATP present and inversely correlates with the amount of kinase activity. The reference compound was diluted as described. The compound was diluted with water, and 5 μl of the dilution was added to 50 μl of the reaction mixture. All enzymatic reactions were carried out at 30°C for 45 minutes. The 50 μl reaction mixture contained 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg / ml BSA, 1 mM DTT, 10 μM ATP, kinase substrate, and the respective enzyme. After the enzymatic reaction, 50 μl of Kinase-Glo Plus Luminescent Kinase Assay Solution (Promega) was added to each reaction mixture, and the plates were incubated at room temperature for 15 minutes. The luminescence signal was measured using a BioTek Synergy 2 microplate reader.

[0163] Data Analysis: Kinase activity assays were performed twice at each concentration. Luminescence data were analyzed using the computer software GraphPad Prism. 100% activity (Lut-Luc) was defined as the difference in luminescence intensity between the absence of kinase (Lut) and the presence of kinase (Luc). Using the luminescence signal in the presence of the compound (Lu), % activity was calculated as follows: % activity = {(Lut-Lu) / (Lut-Luc)} × 100%, where Lu is the luminescence intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table).

[0164] The inhibition results using representative compositions are shown in Tables 67-69 and Figures 24-26. Table 67 and Figure 24 show the results of JAK1 inhibition using representative compositions. Table 68 and Figure 25 show the results of JAK2 inhibition using representative compositions. Table 69 and Figure 26 show the results of JAK3 inhibition using representative compositions. As can be easily seen from these results, the inhibition of all three JAK kinases tested was significant and substantial, consistent with or exceeding the inhibition provided by the reference compounds. JPEG0007884042000074.jpg67166JPEG0007884042000075.jpg65166JPEG0007884042000076.jpg67166

[0165] Tables 70-72 and Figures 27-29 show the corresponding results for the colored fractions. Here, Table 70 shows the results of JAK1 inhibition using the colored fraction of a representative composition. Table 71 shows the results of JAK2 inhibition using the colored fraction of a representative composition, and Table 72 shows the results of JAK3 inhibition using the colored fraction of a representative composition. Table 73 is a table summarizing the results from Tables 70-72. In particular, the synergistic effect on inhibition of all three JAK kinases was observed at high concentrations when all colored fractions were used together in the representative composition, compared to the individual colored fractions. Furthermore, it should be noted again that the compositions presented herein had similar inhibitory properties compared to the reference compound. JPEG0007884042000077.jpg113166JPEG0007884042000078.jpg114166JPEG0007884042000079.jpg113166JPEG0007884042000080.jpg102166 CD39:

[0166] In further experiments, the inventors attempted to determine whether representative compositions affect the enzyme activity of recombinant human CD39 using in vitro enzyme assays.

[0167] The reagents used are shown in Tables 74-77 below, and the tests were performed as described unless otherwise specified (*POM-1 was used as the reference compound). Table 74 shows typical compositions at standard concentrations, and Table 75 shows typical compositions at low concentrations. Table 76 shows the colored fractions of typical compositions at standard concentrations. Here, D5 is the yellow blend, D9 is the purple blend, D21 is the green blend, D13 is the red blend, and D31 is CBD. As mentioned above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. Table 77 shows CD39. JPEG0007884042000081.jpg35166JPEG0007884042000082.jpg39166JPEG0007884042000083.jpg51166JPEG0007884042000084.jpg25166

[0168] Assay conditions: In general, all assay points were performed according to the protocols of the CD39 and CD73 inhibitor screening assay kits (BPS Bioscience, #79278 and 72055, respectively). The CD39 enzymatic reaction was performed twice at room temperature for 30 minutes in a 50 μl mixture containing assay buffer, ATP, CD39 enzyme, and the test compound. The test compound was pre-incubated with the enzyme for 30 minutes. The reaction was initiated by adding the substrate. The 50 μl reaction was performed in a 96-well clear plate. After the enzymatic reaction, 100 μl of colorimetric detection reagent was added to the reaction mixture. After 15 minutes of incubation, absorbance was measured at 630 nm using a Tecan plate reader.

[0169] Data Analysis: Enzyme activity assays were performed twice at each concentration. Absorbance data were analyzed and compared. In the absence of the compound, the intensity of each dataset was defined as 100% (Ce) activity. In the absence of the enzyme, the intensity of each dataset was defined as 0% (C0) activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (Ce - C0), where C is the intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table).

[0170] As shown in Tables 77-78 below, significantly high inhibitory activity was observed for CD39 across all test concentrations. Table 78 and Figure 27 show the results for standard concentrations, while Table 79 and Figure 28 show the results for low concentrations. As is readily apparent from the results, inhibition against the reference inhibitor was unexpectedly strong against the known reference inhibitor POM-1. In particular, the IC50 concentration of the composition was 0.000044%. When the inhibitory activity of the colored fraction was tested, as can be seen from the results in Table 80 and Figure 29, the inhibitory activity was partially distributed to the selected fraction, but not completely. JPEG0007884042000085.jpg62166JPEG0007884042000086.jpg71166JPEG0007884042000087.jpg121166

[0171] Next, the inventors further investigated whether one or more specific plant materials and their polyphenols were associated with inhibitory activity against CD39. To that end, the inventors tested two components of the red blend, namely apple extract (DCH-IC50X) and pomegranate extract (DCH-IC50Y), in a low concentration range as shown in Table 81, under otherwise identical assay conditions. The results are shown in Table 82 and Figure 30. JPEG0007884042000088.jpg49166JPEG0007884042000089.jpg96166

[0172] In further experiments, the inventors also investigated whether CD39 could be inhibited by a multivitamin mixture. To this end, comparative experiments were conducted using the same CD39 experimental procedure as described above, between a multivitamin mixture (labeled DCH-TIV-1.0 (Centrum Multivitamin for Adults)) and a representative composition (labeled DCH-TIC-0.5). The compositions are shown in Table 83. JPEG0007884042000090.jpg43166

[0173] The results of this comparison are shown in Table 84 and Figure 31. As is clear from the results, the representative composition had a very strong inhibitory effect on CD39, while the multivitamin composition had virtually no significant inhibitory effect. JPEG0007884042000091.jpg81166 CD73:

[0174] In further experiments, the inventors attempted to determine whether representative compositions affect the enzyme activity of recombinant human CD73 using in vitro enzyme assays.

[0175] The reagents used are shown in Tables 85-89 below, and unless otherwise specified, the tests were performed as described (*AMPCP or quercetin was used as the reference compound). Table 85 shows typical compositions at standard concentrations, and Table 86 shows the colored fractions of typical compositions at standard concentrations. Here, D5 is the yellow blend, D9 is the purple blend, D21 is the green blend, D13 is the red blend, and D31 is CBD. As mentioned above, the red blend contained apple extract, pomegranate extract, tomato powder, and beet; the green blend contained olive extract, rosemary extract, green coffee bean extract, and kale; the orange-yellow blend contained onion extract, ginger extract, grapefruit extract, and carrot; and the purple-blue blend contained grape extract, blueberry extract, currant, and elderberry. Table 87 shows CD73. JPEG0007884042000092.jpg33166JPEG0007884042000093.jpg47166JPEG0007884042000094.jpg25166

[0176] Assay conditions: In general, all assay points were performed according to the protocols of the CD39 and CD73 inhibitor screening assay kits (BPS Bioscience, #79278 and 72055, respectively). The CD73 enzyme reaction was performed twice at room temperature for 30 minutes in a 50 μl mixture containing assay buffer, ATP, CD73 enzyme, and the test compound. The test compound was pre-incubated with the enzyme for 30 minutes. The reaction was initiated by adding the substrate. The 50 μl reaction was performed in a 96-well clear plate. After the enzymatic reaction, 100 μl of colorimetric detection reagent was added to the reaction mixture. After 15 minutes of incubation, absorbance was measured at 630 nm using a Tecan plate reader.

[0177] Data Analysis: Enzyme activity assays were performed twice at each concentration. Absorbance data were analyzed and compared. In the absence of the compound, the intensity of each dataset was defined as 100% (Ce) activity. In the absence of the enzyme, the intensity of each dataset was defined as 0% (C0) activity. The percentage activity in the presence of each compound was calculated according to the following formula: % activity = (C - C0) / (Ce - C0), where C is the intensity in the presence of the compound (all percentage activities less than zero are shown as zero in the table).

[0178] As can be seen from the results in Tables 88-90, the inhibition of CD73 by the representative compositions and their fractions was remarkably high, especially compared to the current reference standard. Table 88 and Figure 32 show the results of CD73 inhibition at standard concentrations, while Table 89 and Figure 33 show the results of CD73 inhibition at low concentrations. Here, the IC50 of the representative compositions is approximately 0.000044%. Furthermore, as can be seen from these results and the results of the colored fractions shown in Table 90 and Figure 34, a strong synergistic effect of the colored fractions was observed with respect to CD73 inhibition when used in combination (as with the representative compositions). JPEG0007884042000095.jpg61166JPEG0007884042000096.jpg72166JPEG0007884042000097.jpg123166

[0179] To further investigate whether CD73 can also be inhibited by multivitamin formulations, the inventors conducted comparative experiments between a representative composition and a multivitamin composition using the same test procedure outlined above. Table 91 shows the reagents used in this experiment (DCH-TIV-0.5 represents the representative composition, and DCH-TIV-1.0 represents the adult centrum multivitamin). JPEG0007884042000098.jpg45166

[0180] As can be easily seen from the results in Table 92 and Figure 35, the representative compositions exhibited substantial inhibitory activity with respect to CD73, while only slight inhibitory activity was observed in the multivitamin compositions. JPEG0007884042000099.jpg81166

[0181] In some embodiments, numerical values ​​representing properties such as the amount and concentration of components, reaction conditions, etc., used to describe and claim specific embodiments of the present invention should be understood to be modified in some cases by the term “approximately.” Therefore, in some embodiments, numerical parameters shown in the written description and appended claims are approximations that may vary depending on the desired properties to be obtained by the particular embodiment. The enumeration of value ranges herein is merely intended to serve as a simple way to individually refer to individual values ​​falling within that range. Unless otherwise indicated herein, individual values ​​are incorporated into the specification as if they were individually enumerated herein.

[0182] As used herein, the term “administer” a pharmaceutical composition or dietary supplement composition means both direct and indirect administration of the pharmaceutical composition or dietary supplement composition, where direct administration of the pharmaceutical composition or dietary supplement composition is typically performed by a healthcare professional (e.g., a physician, nurse, dietitian, etc.), and indirect administration includes the step of providing or making available the pharmaceutical composition or dietary supplement composition to a healthcare professional or individual who needs it for direct administration (e.g., by injection, infusion, oral delivery, topical delivery, etc.). Furthermore, it should be noted that the term “predict” a condition, susceptibility to the onset of a disease, or response to an intended treatment means covering acts or predictions (but not treatment or diagnosis) that predict a condition, susceptibility and / or response, including the progression, improvement and / or duration of the condition in question.

[0183] All methods described herein may be carried out in any suitable order, unless otherwise indicated herein or unless it is clearly inconsistent with the context. The use of any examples or illustrative words (e.g., "etc.") provided in reference to specific embodiments herein is intended merely to better illustrate the invention and not to limit the scope of the invention as otherwise claimed. Wording in this specification should not be construed as indicating unclaimed elements essential to the carrying out of the invention.

[0184] As used herein and throughout the claims, the meanings of “a,” “an,” and “the” include multiple references unless the context explicitly indicates otherwise. Also, as used herein, the meaning of “in” includes “in” and “above” unless the context explicitly indicates otherwise. Also, as used herein, unless the context explicitly indicates otherwise, the term “joined” is intended to include both direct joining (two elements joined together are in contact with each other) and indirect joining (at least one additional element is in contact with each other). Thus, the terms “joined to” and “joined with” are used as synonyms.

[0185] Those skilled in the art will see that many modifications beyond those already described are possible without departing from the concept of the present invention. Therefore, the subject matter of the present invention should not be limited beyond the appended claims. Furthermore, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible way that is consistent with the context. In particular, the term “including” should be interpreted to mean an element, component, or step that is present, utilized, or may be combined with other elements, components, or steps that are not expressly referenced. Where the specification or claims refer to at least one selected from the group consisting of A, B, C… and N, the description should be interpreted as requiring only one element from the group, rather than A and N or B and N, etc.

Claims

1. A composition for inhibiting one or more enzymes involved in the pathways of health and healthy aging, wherein the enzyme is selected from the group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3, and the composition comprises chemically different polyphenols. The polyphenols include organic acids, phenols, flavonols, flavanols, anthocyanins, chlorogenic acid, and betacyanins. The polyphenols are derived from plant materials having red, green, orange-yellow, and purplish-blue colors, wherein the plant material having red is selected from the group consisting of apple extract, pomegranate extract, tomato powder, and beet root powder; the plant material having green is selected from the group consisting of olive extract, rosemary extract, green coffee bean extract, and kale powder; the plant material having orange-yellow is selected from the group consisting of onion extract, ginger extract, grapefruit extract, and carrot powder; and the plant material having purplish-blue is selected from the group consisting of grape extract, blueberry extract, currant powder, and elderberry powder, and The composition is characterized in that the polyphenols are in a synergistic combination with respect to the inhibition of at least one biochemical marker selected from the group consisting of BACE1, CD38, CD73, CDK5, JAK1, JAK2, and JAK3.

2. The composition according to claim 1, wherein the polyphenol is provided in the form of the plant material.

3. The composition according to claim 1, wherein the composition is administered in vivo.

4. The composition according to claim 1, wherein the composition is administered to a mammal.

5. The composition according to claim 1, wherein the composition provides immune support, metabolic support, longevity support, central nervous system (CNS) function support, reduction of inflammatory responses, reduction of the effects of cardiovascular disease, and reduction of amyloid-beta plaque formation.

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